Antibiotic Resistance Pattern and Evaluation of blaOXA-10, blaPER-1, blaVEB, blaSHV Genes in Clinical Isolates of Pseudomonas aeruginosa Isolated from Hospital in South of Iran in 2014-2015

Antibiotic Resistance Pattern and Evaluation of blaOXA-10, blaPER-1, blaVEB, blaSHV Genes in Clinical Isolates of Pseudomonas aeruginosa Isolated from Hospital in South of Iran in 2014-2015

The extended-spectrum-beta-lactamase (ESBL) producing Gram-negative bacteria (GNB) has been implicated in the global spread of multi-drug resistance (MDR) genes leading to limited therapeutic options. This study aimed to determine the frequency of blaCTX-M and blaSHV ESBL genes in clinical isolates from patients with GNB infection in Akwa Ibom State, Nigeria. A cross-sectional study of patients having various infections was conducted at the University of Uyo Teaching Hospital, Uyo. Clinical samples were cultured by standard bacteriological methods and isolates identified using VITEK-2 protocols. Gram-negative bacteria identified were screened for antibiotics sensitivity, ESBL production and possession of ESBL genes using Kirby-Bauer disc diffusion, double disc synergy test and polymerase chain reaction, respectively. Out of 180 clinical samples of urine, blood and wound, 71 consecutive non-repetitive GNB were isolated of which 29% were ESBL producers. The GNB recovered from the samples were 35 (58.3%), 22 (36.7%) and 14 (23.3%), of which 12 (34.3%), 9 (40.9%) and 8 (57.1%), were ESBL producers, respectively. Escherichia coli was the most prevalent GNB and the highest ESBL producer (14.1%). Susceptibility test showed moderately high resistance of GNB to trimethoprim-sulfamethoxazole (59.1%), ceftazidime (56.3%) and cefotaxime (54.9%). Of the selected 25 ESBL-producers, 15 (60%) possessed the blaCTX-M genes while one (4%) harboured the blaSHV gene. The blaCTX-M detection rates in wound, blood and urine were 24%, 20% and 16%, respectively. Isolates with the blaCTX-M genes were E. coli, S. fonticola, K. pneumoniae, P. mirabilis, A. baumannii, K. oxytoca, B. cepacia, E. cloacae, and P. aeruginosa while Serratia fonticola carried the blaSHV gene. The implication of blaCTX-M genes in MDR could be associated with treatment failures in patients with GNB infections. Antimicrobial stewardship to guide appropriate and prudent use of antibiotics is advocated.

BackgroundThe iron/siderophore uptake system (IUS) involved in the Acinetobacter baumannii pathogenicity. However, IUS's role in antibiotic resistance and the production of β-lactamase enzymes of A. baumannii are unclear. This study aimed to investigate the relationship between the production of β-lactamase enzymes and IUS regulatory genes in clinical isolates of A. baumannii. Methods. A. baumannii isolates were collected from clinical isolates using biochemical tests. The antibiotic resistance patterns and β-lactamase-producing strains were identified using the disk diffusion method (DDM). Also, IUS genes were detected by the polymerase chain reaction (PCR) method. Results Seventy-two (72) A. baumannii isolates were collected from a different clinical specimen. Gentamicin-resistant strains (43%) had the highest frequency, and aztreonam-resistant strains (12.5%) had the lowest frequency. Also, the distribution of AmpC and MBL producing isolates were 27.7% and 35%, respectively. Moreover, the frequencies of basD, bauA, pld, paaE, entA, feoB, hemO, and tonB genes were as follows: 12.5%, 15.2%, 11.1%, 15.2%, 19.4%, 16.6%, 23.6%, and 6.9%. Further, a strong correlation was observed between the abundance of β-lactamase-producing strains and IUS genes. Conclusions Based on our knowledge from this study, the association between β-lactamase production and IUS genes in A. baumannii plays an essential role in the emergence of drug-resistant strains.

The diversity and distribution of TEM, SHV and CTX-M type of extended-spectrum beta-lactamases (ESBLs) are important for the treatment and control of infections. Determination of ESBL genes in clinical isolates by polymerase chain reaction (PCR) and DNA sequencing can obtain useful data for their molecular epidemiology and risk. The aim of this study was to investigate the frequency of beta-lactamase genes in Acinetobacter baumannii strains isolated from different regions of Turkey. A total of 519 A.baumannii strains collected from hospitals located at 12 different provinces of Turkey (Bolu (n= 67), Tokat (n= 47), Trabzon (n= 25), Ordu (n= 27), Diyarbakır (n= 47), Niğde (n=31), Kayseri (n= 36), Ankara (n= 41), Kirikkale (n= 26), Kahramanmaraş (n= 25), Mersin (n= 40), Istanbul (n= 107)] between 2011-2012 period were included in the study. Identification of the isolates were performed by both conventional methods and automated systems, VITEK2 Compact (BioMerieux, France) and API 32GN (BioMerieux, France). Disc diffusion method was used for the detection of antibiotic susceptibilities of the isolates and the results were evaluated according to CLSI (Clinical and Laboratory Standards Institute) criteria. Tigecycline and colistin sensitivities of the isolates were evaluated according to BSAC (British Society for Antimicrobial Chemotherapy) criteria. The presence of beta-lactamase genes, namely blaoxa-51, blaTEM, blaSHV, blaCTX-M1, blaCTX-M2, blaGES and blaVIM were detected by PCR. In our study, the resistance rates against colistin, tigecycline, ampicillin-sulbactam, amoxicillin-clavulanic acid, cefoperazone/sulbactam, tobramycin, ceftriaxone, piperacillin-tazobactam, gentamicin, ampicillin, tetracycline, cefepime, piperacillin, amikacin, trimethoprim-sulfamethoxazole, meropenem, levofloxacin, ciprofloxacin, imipenem and ceftazidime were detected as; 0.6%, 2.7%, 11.9%, 15.2%, 21%, 22.9%, 23.9%, 48.6%, 59.5%, 61.8%, 66.3%, 67.8%, 69.2%, 71.1%, 77.5%, 78.6%, 81.1%, 82.9%, 87.5% and 89.4%, respectively. All of the isolates (100%) were OXA-51 positive, while 443 (85.4%) out of 519 strains harbored other beta-lactamase genes searched in the study. When the distribution of the genes were evaluated, blaTEM-1 was found as the predominant one with a frequency rate of 55.7% (n=289/519), followed by blaCTX-M2 (63/519, 12.1%), blaCTX-M1 (42/519, 8.1%), blaSHV (40/519, 7.7%), blaGES (8/519, 1.5%) and blaVIM (1/519, 0.2%). Cooccurence of ESBL genes was detected in 16.3% (72/443) of the strains, being mostly TEM+CTX-M2 (20/72, 27.8%), TEM+SHV (11/72, 15.3%) and TEM+CTX-M1 (10/72, 13.9%). In addition, it was noted that the distribution of ESBL genes between isolates showed differences according to the provinces. Accordingly, none of the strains isolated from four provinces (Bolu, Niğde, Mersin, Kahramanmaraş) and from three provinces (Bolu, Kahramanmaraş, Diyarbakir) harbored blaCTX-M1/M2 and blaSHV genes, respectively. The blaTEM gene was detected in isolates collected from all of the provinces, with a highest frequency in Niğde (28/31, 90.3%) and lowest in Trabzon (1/25, 4%). The presence of GES-11 type ESBLs was found only in the isolates sent from Niğde province (8/31; 25.8%). Screening of metallo-beta-lactamase VIM gene also yielded a single positive result amongst only Niğde isolates (1/31; 3.2%), and this gene was identified as VIM-5 type by DNA sequencing. This study which is the first comprehensive national research to characterize ESBLs in A.baumannii isolates by molecular methods, showed that the most prevalent ESBL type is TEM (289/519, 55.7%) amongst A.baumannii strains isolated from different regions of our country. The data of our study is parallel to the results of previous studies carried out from Turkey.

The type III secretion system of Pseudomonas aeruginosa transports four known effector proteins: ExoS, ExoT, ExoU and ExoY. However, the prevalence of the type III secretion system genes or the effector-encoding genes in clinical and environmental isolates of P. aeruginosa has not been well studied. Southern hybridization analyses and PCR were performed on over 100 P. aeruginosa isolates to determine the distribution of these genes. Clinical isolates were obtained from urine, endotracheal, blood and wound specimens, from the sputum of cystic fibrosis (CF) patients, and from non-hospital environmental sites. The popB gene was used as a marker for the presence of the large chromosomal locus encoding the type III secretion machinery proteins. Each isolate contained the popB gene, indicating that at least a portion of this large chromosomal locus was present in all isolates. Likewise, each isolate contained exoT-like sequences. In contrast, the exoS, exoU and exoY genes were variable traits. Overall, 72% of examined isolates contained the exoS gene, 28% contained the exoU gene, and 89% contained the exoY gene. Interestingly, an inverse correlation was noted between the presence of the exoS and exoU genes in that all isolates except two contained either exoS or exoU but not both. No significant difference in exoS, exoU or exoY prevalence was observed between clinical and environmental isolates or between isolates cultured from different disease sites except for CF respiratory isolates. CF isolates harboured the exoU gene less frequently and the exoS gene more frequently than did isolates from some of the other sites of infection, including the respiratory tract of patients without CF. These results suggest that the P. aeruginosa type III secretion system is present in nearly all clinical and environmental isolates but that individual isolates and populations of isolates from distinct disease sites differ in their effector genotypes. The ubiquity of type III secretion genes in clinical isolates is consistent with an important role for this system in human disease.

BackgroundThe aminoglycoside-resistance genes encoding aminoglycoside modifying enzymes and 16S rRNA methyltransferases are main factors contributing to increasing resistance to aminoglycosides. Characterization and distribution of antimicrobial resistance gene profiles provide important information on the potential difficulty of treatment of bacteria. Several molecular methods have been developed to investigate the prevalence of aminoglycoside-resistance genes. These existing methods are time-consuming, labor-intensive, expensive or limited sensitivity in the epidemiological investigation. Therefore, it is necessary to develop a rapid, less-costly and high throughput and sensitive method to investigate the distribution of antimicrobial resistance gene in clinical isolates.ResultsIn this study, we developed a GeXP analyzer-based multiplex PCR assay to simultaneously detect seven aminoglycoside-resistance genes, including aac(3)-II, aac(6′)-Ib, aac(6′)-II, ant(3″)-I,aph(3′)-VI,armA and rmtB, and to analyze the distribution of these genes in clinical Enterobacteriaceae isolates. Under optimized conditions, this assay achieved a limit-of-detection as low as 10 copies of each of the seven genes. The presented method was applied to analyze the distribution of aminoglycoside-resistance genes in 56 clinical Enterobacteriaceae isolates, and the results were compared with that of the conventional single PCR assay. Kappa values of the two methods for detecting each of the seven resistance genes were 0.831, 0.846, 0.810, 0.909, 0.887, 0.810 and 0.825, respectively.ConclusionThis GeXP assay is demonstrated to be a rapid, cost-effective and high throughput method with high sensitivity and specificity for simultaneously detecting seven common aminoglycoside-resistance genes.

Expression of multidrug resistance efflux pump genes in clinical and environmental isolates of Staphylococcus aureus

Antimicrobial resistance patterns and their encoding genes among clinical isolates of Acinetobacter baumannii in Ahvaz, Southwest Iran

Pseudomonas aeruginosa is a significant nosocomial pathogen notorious for its multidrug resistance (MDR), often mediated by efflux pumps like MexAB-OprM. This study aimed to molecularly identify the mexB gene within clinical isolates of P. aeruginosa and assess its correlation with antibiotic resistance profiles. This study aims to detect the presence of the MexB efflux pump gene in clinical isolates of Pseudomonas aeruginosa and to investigate its association with antibiotic resistance patterns, particularly multidrug resistance, to better understand the molecular mechanisms underlying antimicrobial resistance and support effective therapeutic strategies. Methodology: All clinical isolates of P. aeruginosa from patients at Teaching Ba'aqubah Hospital and Teaching Albatool Hospital were investigated in this study. identified with the Vitek 2 compact, MexB efflux pump genes are detected by the PCR technique and primers are designed in the biology department of the College of Education of Pure Science in Iraq. Conventional PCR results showed that the gene, MexB, was present in all seven clinical isolates of P. aeruginosa. It was concluded that carrying efflux pump genes can make P. aeruginosa more resistant to several drugs. Result: From the 100 clinical specimens collected P. aeruginosa recorded a high percentage of isolated 67(67%), P. aeruginosa isolates Including; Chronic burns swab 23 (34.3%), Wounds swab 15 (22.4%), Urine from UTI patients 12 (17.9%), Sputum 10 (14.9%) and Ear swabs 7 (10.5%). All isolates were obtained by cultural characterisation These clinical isolates were on cetrimide agar, which is a selective medium for Pseudomonas spp.at 37°C for 24 hours. the VITEK2 compact System to confirm the diagnosis of Pseudomonas aeruginosa. Conclusion: According to our research, any P. aeruginosa that has the MexB gene of the efflux pump in the MexABOprM operon may enhance drug expulsion and antibiotic resistance, which could cause health issues for patients, particularly those with compromised immune systems.

Mycobacterium tuberculosis growth rate is closely coupled to rRNA transcription which is regulated through carD gene. The aim of this study was to determine the sequence of carD gene in drug susceptible and resistant clinical isolates of M. tuberculosis and designing of a PCR assay based on carD sequence for rapid detection of this bacterium.Specific primers for amplification of carD gene were carefully designed, so that whole sequence of gene could be amplified; therefore primers were positioned at the upstream (promoter of this gene and ispD gene) and downstream (in ispD gene). DNA from 41 clinical isolates of M. tuberculosis with different pattern of drug resistance was used in the study. PCR conditions and annealing temperature were designed by means of online programs. PCR products were sequenced by ABI system.PCR product of carD gene was a 524 bp fragment. This method could detect all resistant and susceptible strains of M. tuberculosis. The size of amplified fragment was similar in all investigated samples. Sequence analysis showed that there was similar sequence in all of our isolates therefore probably this gene is considered to be conservative. Translation of nucleotide mode to amino acids was showed that TRCF domain in N-terminal of protein CarD was found to be fully conservative.This is the first study on the sequence of carD gene in clinical isolates of M. tuberculosis. This conservative gene is recommended for use as a target for designing of suitable inhibitors as anti-tuberculosis drug because its importance for life of MTB. In the other hand, a PCR detection method based on detection of carD gene was recommended for rapid detection in routine test.

Background: Pseudomonas aeruginosa is one of the most important causes of opportunistic infections. Carbapenems are administrated for the treatment of resistant infections due to this bacterium. The aim of this study was to investigate the presence of blaSHV, blaTEM, blaCTX-M and blaOXA-48 beta-lactamase genes in clinical isolates of P. aeruginosa in Bandar Abbas, Iran. Methods: In this descriptive study, 96 isolates of P. aeruginosa were obtained from clinical samples in Bandar Abbas. All isolates were identified by biochemical tests. The antibiotic susceptibility was examined by disk diffusion method. The presence of blaCTX-M, blaSHV, blaTEM and blaOXA-48 genes was evaluated by polymerase chain reaction using a specific primer. Results: In this study, 76 isolates of P. aeruginosa (79.2%) were resistant to at least one carbapenem. The highest bacterial resistance (100%) was obtained to nalidixic acid (30 µg), followed by tetracycline (30 µg) and amoxicillin (25 µg). The frequencies of blaCTX-M, blaSHV, blaTEM and blaOXA-48 genes were 23.95% (23 isolates), 23.08% (26 isolates), 57.29% (55 isolates) and 12.5% (12 isolate), respectively. Sixty isolates resistant to carbapenems (78.9%) carried at least one resistant gene (blaTEM, blaSHV, blaCTX-M and blaOXA-48) and 16 isolates (21.1%) did not have any of these genes. Conclusions: This study revealed the prevalence of carbapenem-resistant phenotype of P. aeruginosa clinical isolates and the frequency of extended-spectrum β-lactamase genes have increased.

Anthrax is a zoonotic disease caused by Bacillus anthracis, and well recognized as a potential agent for bioterrorism. B. anthracis can be identified by detecting the virulence factors genes located on two plasmids, pXO1 and pXO2. The aim of the present study was to determine the presence of virulence genes in 27 isolates of B. anthracis isolated from clinical and environmental samples. For this purpose, multiplex PCR and DNA probes were designed to detect protective antigen ( pag), edema factor (cya), lethal factor (lef ), and capsule (cap) genes. Our results indicated that all the isolates contained all the above virulence genes, suggesting that the isolates were virulent. To the best our knowledge, this is the first study about the determination of virulence marker genes in clinical and environmental isolates of B. anthracis using multiplex PCR and DNA probes in India. We suggest that the above methods can be useful in specific identification of virulent B. anthracis in clinical and environmental samples.

The Mycobacterium tuberculosis genome contains four highly related genes which present significant similarity to Pseudomonas aeruginosa genes encoding phospholipase C enzymes. Three of these genes, plcA, plcB and plcC, are organized in tandem (locus plcABC). The fourth gene, plcD, is located in a different region. This study investigates variations in plcABC and plcD genes in clinical isolates of M. tuberculosis, Mycobacterium africanum and 'Mycobacterium canettii'. Genetic polymorphisms were examined by PCR, Southern blot hybridization, sequence analysis and RT-PCR. Seven M. tuberculosis isolates contain insertions of IS6110 elements within plcA, plcC or plcD. In 19 of 25 M. tuberculosis isolates examined, genomic deletions were identified, resulting in loss of parts of genes or complete genes from the plcABC and/or plcD loci. Partial plcD deletion was observed in one M. africanum isolate. In each case, deletions were associated with the presence of a copy of the IS6110 element and in all occurrences IS6110 was transposed in the same orientation. A mechanism of deletion resulting from homologous recombination of two copies of IS6110 was recognized in a group of genetically related M. tuberculosis isolates. Five M. tuberculosis isolates presented major polymorphisms in the plcABC and plcD regions, along with loss of expression competence that affected all four plc genes. Phospholipase C is a well-known bacterial virulence factor. The precise role of phospholipase C in the pathogenicity of M. tuberculosis is unknown, but considering the potential importance that the plc genes may have in the virulence of the tubercle bacillus, the study of isolates cultured from patients with active tuberculosis bearing genetic variations affecting these genes may provide insights into the significance of phospholipase C enzymes for tuberculosis pathogenicity.

In view of the significant negative impact of biofilm mediated infection on patient health and the necessity of a reliable phenotypic method for detecting biofilm producers, this study aimed to determine biofilm producing ability and presence of icaAD gene in clinical staphylococcal isolates as well as to assess the reliability of two phenotypic methods used for detection of biofilm. A total of 50 staphylococcal strains were isolated from 124 clinical specimen (94 intravascular catheters and 30 blood samples) collected from in-patients at Pediatric Hospital of Ain Shams University. Two phenotypic methods were used for detection of biofilm production; qualitative Congo red agar (CRA) and quantitative Microtiter plate (MTP). PCR was used to determine the presence of icaAD gene. Biofilm production was detected in 23(46%) isolates by CRA and MTP, however, both methods correlated only in 10(20%) of isolates. The icaAD gene was detected in 16(32%) staphylococcal isolates. Correlating phenotypic methods with icaAD gene detection, only 8(50%) of the icaAD positive staphylococci were positive by MTP, while 5(31%) were positive by CRA method. Unexpectedly, 15(30%) and 18 (36%) of the isolates were icaAD negative while MTP and CRA positive, respectively.In conclusion, despite the presence of icaAD gene, it does not always correlate with in vitro biofilm formation. The biofilm-forming ability of some isolates in absence of icaAD gene highlights the importance of further genetic investigations of ica independent biofilm formation mechanisms. Comparing phenotypic methods, MTP remains a better tool for biofilm screening.

In this study two control isolates of Salmonella enteritidis, RTCC1623 and RTCC1624, were obtained from the institute ofRazi (Karaj-Iran) and 14 strains were isolated from poultry samples in Kermanshah province of Iran, according to a standard protocol. These isolates were confirmed by PCR amplification of SefA gene fragments. Results showed that, 6 isolates of 14 isolates of Salmonella which their biochemical tests were positive contain 511 bp amplified fragments of the SefA gene. In other purpose, to correlating the presence of plasmids with antibiotic resistance and protein pattern, plasmid DNA was isolated before and after plasmid curing by using the alkaline lysis method. Strains of S. enteritidis contain seven different plasmid profiles (P1-P7) which were characterized by antibiotic resistance and protein pattern. Our observed showed, there was a high molecular weight plasmid with Rf 0.17 in all strains and the frequency of other plasmids was low. The plasmid with Rf about 0.2 is responsible for resistance to Cephalothin and the isolates that lost it were susceptible to this antibiotic. All strains, 100%, were resistant to ampicillin before and after curing of strains. According to present findings, PCR is a rapid and sensitive method for typing of S. enteritidis and plasmid profiling; antibiotic resistance and protein pattern are suitable methods for subtyping of S. enteritidis isolates. No direct correlation was found between plasmid contents, antibiotic resistance patterns and protein profiles of local S. enteritidis isolates.