Mohammed Ali M. Marie

Prevalence of 16S rRNA methylase genes among β-lactamase-producing Enterobacteriaceae clinical isolates in Saudi Arabia.

Background Co production of 16S rRNA methylases gene and β-Lactamase gene among Enterobacteriaceae isolates conferring resistance to both therapeutic options has serious implications for clinicians worldwide. Methods To study co existence of 16S rRNA methylases (armA, rmtA, rmtB, rmtC, rmtD, and npmA) and β-Lactamase (blaTEM-1, blaSHV-12, blaCTX-M-14) genes, we screened all phenotypic positive β-Lactamase producing enterobacteriaceae by polymerase chain reaction (PCR) targeting above genes. A total of 330 enterobacteriaceae strains were collected during study period out of that 218 isolates were identified phenotypically as β-Lactamase producers, which include 50 (22.9%) Escherichia coli; 92 (42.2%) Klebsiella pneumoniae, 44 (20.2%), Citrobactor freundii and 32 (14.7%) Enterobacter spp. Results Among this 218, only 188 isolates harbored the resistant gene for β-Lactamase production. Major β-Lactamase producing isolates were blaTEM-1 type. 122 (56 %) isolates were found to produce any one of the 16S rRNA methylase genes. A total of 116 isolates co produced β-Lactamase and at least one 16S rRNA methylases gene Co production of armA gene was found in 26 isolates with rmtB and in 4 isolates with rmtC. The rmtA and rmtD genes were not detected in any of the tested isolates. Six isolates were positive for a 16S rRNA methylase gene alone. Conclusion β-Lactamase producing isolates appears to coexist with 16S rRNA methylase predominantly armA and rmtB genes in the same isolate. We conclude the major β-Lactamase and 16S rRNA methylases co-producer was K. pneumoniae followed by E. coli. We suggest further work on evaluating other β-lactamases types and novel antibiotic resistance mechanisms among Enterobacteriaceae.Background Co production of 16S rRNA methylases gene and β-Lactamase gene among Enterobacteriaceae isolates conferring resistance to both therapeutic options has serious implications for clinicians worldwide. Methods To study co existence of 16S rRNA methylases (armA, rmtA, rmtB, rmtC, rmtD, and npmA) and β-Lactamase (blaTEM-1, blaSHV-12, blaCTX-M-14) genes, we screened all phenotypic positive β-Lactamase producing enterobacteriaceae by polymerase chain reaction (PCR) targeting above genes. A total of 330 enterobacteriaceae strains were collected during study period out of that 218 isolates were identified phenotypically as β-Lactamase producers, which include 50 (22.9%) Escherichia coli; 92 (42.2%) Klebsiella pneumoniae, 44 (20.2%), Citrobactor freundii and 32 (14.7%) Enterobacter spp. Results Among this 218, only 188 isolates harbored the resistant gene for β-Lactamase production. Major β-Lactamase producing isolates were blaTEM-1 type. 122 (56 %) isolates were found to produce any one of the 16S rRNA methylase genes. A total of 116 isolates co produced β-Lactamase and at least one 16S rRNA methylases gene Co production of armA gene was found in 26 isolates with rmtB and in 4 isolates with rmtC. The rmtA and rmtD genes were not detected in any of the tested isolates. Six isolates were positive for a 16S rRNA methylase gene alone. Conclusion β-Lactamase producing isolates appears to coexist with 16S rRNA methylase predominantly armA and rmtB genes in the same isolate. We conclude the major β-Lactamase and 16S rRNA methylases co-producer was K. pneumoniae followed by E. coli. We suggest further work on evaluating other β-lactamases types and novel antibiotic resistance mechanisms among Enterobacteriaceae.

Molecular characterization of the β-lactamases in Escherichia coli and Klebsiella pneumoniae from a tertiary care hospital in Riyadh, Saudi Arabia

The widespread use of antimicrobials has increased the occurrence of multidrug resistant microbes. The commonest mechanism of antimicrobial resistance in Enterobacteriaceae is production of β‐lactamases such as metallo‐β‐lactamases (MBL) and extended spectrum β‐lactamases (ESBL). Few studies have used a molecular approach to characterize the prevalence of β‐lactamases. Here, the prevalence of different β‐lactamases was characterized by performing three multiplex PCRs targeting genes similar to those described in earlier publications. Antimicrobial susceptibility tests for all isolates were performed using the agar dilution method. β‐lactamase was detected in 72% of the isolates, the detection rate being 64% in 2011 and 75% in 2012. The isolates were highly resistant to carbapenems such as meropenem and imipenem and susceptible to colistin and tigecycline. In this study, 22% of isolates contained both MBL and ESBL. ESBL was detected more frequently in Escherichia coli isolates, whereas carbapenemase was detected more frequently in Klebsiella pneumoniae isolates. These findings suggest the spread of multi‐resistant ESBL and MBL producers in the community. Our results have implications for patient treatment and also indicate the need for increased surveillance and molecular characterization of isolates.

Characterization of carbapenem resistance mechanisms in Klebsiella pneumoniae and in vitro synergy of the colistin–meropenem combination

Abstract In this prospective study, consecutive isolates of Klebsiella pneumoniae were tested for different mechanisms of carbapenem resistance using the modified Hodge test (MHT), Rosco Neo-Sensitabs (ROSCO). Phenylalanine arginine beta-naphthylamide assay (PABN) inhibitor-based test was done on isolates in which the mechanism of resistance was not identifiable by the ROSCO. Among 105 selected isolates, carbapenemase production was noted in 100 (95%) by MHT and ROSCO showed 97 (92·4%) inhibition with dipicolinic acid signifying the production of MBL. PCR amplification was positive in 90 (86%) isolates for blaNDM-1 and 46 (44%) isolates for blaOXA-48. 54 (51%) isolates were positive for blaCTX-M and all belonged to blaCTX-M group 1. Isolates co produced blaOXA-48 (31/105, 30%) and blaCTX-M (40/105, 38%) in combination with the carbapenemase (blaNDM-1) gene. Five colistin-resistant isolates were positive for blaOXA-48. Eight isolates did not show inhibition with any of the inhibitor containing disks and found to be positive for blaOXA-48. Isolates were tested for colistin-meropenem synergy and detection rate was higher by the checkerboard (48%) than E-test method (35%). Our study necessitates continuous surveillance to recognize the predominant machinery of resistance in a particular geographical region to formulate effective control measures.

Relationship between Helicobacter pylori virulence genes and clinical outcomes in Saudi patients.

Helicobacter pylori has been strongly associated with gastritis, gastric and duodenal ulcers, and it is a risk factor for gastric cancer. Two major virulence factors of H. pylori have been described: the cytotoxin-associated gene product (cagA) and the vacuolating toxin (vacA). Since considerable geographic diversity in the prevalence of H. pylori virulence factors has been reported, the aim of this work was to determine if there is a significant correlation between different H. pylori virulence genes (cagA and vacA) in 68 patients, from Saudi Arabia, and gastric clinical outcomes. H. pylor was recognized in cultures of gastric biopsies. vacA and cagA genes were detected by polymerase chain reaction (PCR). The cagA gene was obtained with 42 isolates (61.8%). The vacA s- and m- region genotypes were determined in all strains studied. Three genotypes were found: s1/m1 (28%), s1/m2 (40%) and s2/m2 (26%). The s2/m1 genotype was not found in this study. The relation of the presence of cagA and the development of cases to gastritis and ulcer was statistically significant (P < 0.05). The study showed a significant correlation between the vacA s1/m2 genotype and gastritis cases, and a significant correlation between vacA s1/m1 genotype and peptic ulcer cases. The results of this study might be used for the identification of high-risk patients who are infected by vacA s1/m1 genotype of H. pylori strains. In conclusion, H. pylori strains of vacA type s1 and the combination of s1/m1 were associated with peptic ulceration and the presence of cagA gene.

Serological and molecular capsular typing, antibiotic susceptibility of Streptococcus pneumoniae isolates from invasive and non-invasive infections

Streptococcus pneumoniae causes life threatening infections and necessitate for impediment and controlling disease; to conquer this, information is needed about serotype distribution and patterns of antibiotic resistance. The present study was to determine the serotype distribution of S. pneumoniae isolated from the entire age group individual and to correlate this distribution with susceptibility. Cases of pneumococcal infections have been reviewed for serotyping and antibiotic susceptibility. Out of 117 pneumococcal isolates 45 (39%) were penicillin-resistant, 84 (72%) were erythromycin-resistant and 100% were co-trimoxazole resistant. The most frequently isolated serotypes were 23F, 19F, 14, 6B, 5, 6A, 19A and 9V. PCV7, PCV10 and PCV13 coverage was 68%, 79%, 87%, respectively. Similarly, there was similarity in PCV7 coverage for non invasive isolates (64.5%) and invasive isolates (72.2%). The study state that common pneumococcal serotypes were present in similar ways as reported in literature. A continuous survey of pneumococcal infected population is requirement and necessity for success of vaccination.

A 6-year surveillance of antimicrobial resistance patterns of Acinetobacter baumannii bacteremia isolates from a tertiary care hospital in Saudi Arabia during 2005-2010

Multidrug resistance (MDR) of Acinetobacter baumannii increasingly jeopardizes the health care setting leading to substantial mortality and morbidity globally. During the past decade, entirely resistant A. baumannii strains presented a real challenge to clinicians and posed difficulties in therapy. MDR of A. baumannii– associated infections, with adverse clinical outcomes involving the respiratory tract, blood, soft tissues, urinary tract, and central nervous system, significantly increases the outlay of the infirmary. Being Gram-negative coccobacilli and an obligate aerobe, A. baumannii causes both community- and hospital-acquired infection outbreaks in intensive care units especially in countries with tropical climates. Of particular concern, we sought to reveal the status of antimicrobial resistance in A. baumannii bacteremia isolates, the trends and relative frequency of multidrug resistance pattern and also the underlying clinical condition among patients with A. baumannii bacteremia at Riyadh Military hospital, Saudi Arabia, from January 2005 to December 2010. (Published: 3 April 2014) Citation: Libyan J Med 2014, 9 : 24039 - http://dx.doi.org/10.3402/ljm.v9.24039

Isolation and characterization of quorum‐sensing signalling molecules in Pseudomonas aeruginosa isolates recovered from nosocomial infections

Pseudomonas aeruginosa is one of the most common pathogens in nosocomial infections. Many studies have documented the role of quorum‐sensing (QS) systems in antibiotic tolerance of P. aeruginosa. N‐acyl homoserine lactones (AHLs) serve as QS signalling molecules and can be a target for modulating bacterial pathogenicity. In this study, nosocomial isolates of P. aeruginosa were characterized for the presence of different types of QS signalling molecules. AHLs were solvent extracted and quantified by determination of β‐galactosidase activity using the Escherichia coli MG4 reporter strain. Further characterization was performed by analytical thin layer chromatography coupled with detection using the Agrobacterium tumefaciens A136 biosensor strain. All P. aeruginosa isolates produced AHLs, but there were differences in the quantity and nature of AHLs. We identified AHLs belonging to C4‐homoserine lactone (HSL), C6‐HSL, C8‐HSL, C10‐HSL and C12‐HSL. AHL profiling of P. aeruginosa isolates showed differences in the amounts and types of AHLs, suggesting differences in the virulence factors and the potential for infection. Our results may be investigated further using animal model systems.

Prevalence and risk factors of early fecal carriage of Enterococcus faecalis and Staphylococcus spp and their antimicrobial resistant patterns among healthy neonates born in a hospital setting in central Saudi Arabia

Objectives: To investigate the prevalence, antibiotic resistant profiles, and risk factors of early fecal carriage of Enterococcus faecalis (E. faecalis) and staphylococci among 150 healthy Saudi neonates born in a hospital setting in central Saudi Arabia. Methods: This prospective study was conducted in Al-Bukayriyah General Hospital, Qassim, Saudi Arabia, between June 2012 and January 2013. The E. faecalis and Staphylococcus spp. isolates were identified manually, and Vitek2 system was used for identity confirmation at the species level and minimum inhibitory concentration-susceptibility testing. Results: Enterococcus faecalis (n=73) and Staphylococcus spp. (n=18) were recovered. Unlike staphylococci, E. faecalis colonization did not significantly vary from day one up to 7 days of life, regardless of the type of feeding, but it was relatively higher among vaginally versus cesarean delivery. Both Staphylococcus epidermidis (S. epidermidis) and Staphylococcus aureus carriage increase as the body weight increases, and this difference was significant (p=0.025) for S. epidermidis. High-level resistance in Gentamycin among E. faecalis isolates was 25% and 11% to Streptomycin. Thirty percent of S. epidermidis were resistant to oxacillin and exhibited multidrug-resistant (MDR) patterns of 5 resistant markers, which were also observed among 2/5 (40%) of Methicillin-resistant Staphylococcus aureus isolates. Conclusion: Enterococcus faecalis did not significantly vary in relation to type of delivery, age up to 7 days, and type of feeding. The neonatal fecal carriage of MDR isolates should be considered as a crucial reservoir to the further spread of antimicrobial resistance genes among hospitals, cross infections, and the community.

A prospective evaluation of synergistic effect of sulbactam and tazobactam combination with meropenem or colistin against multidrug resistant Acinetobacter baumannii

The present study evaluates the synergistic effect of sulbactam/tazobactam in combination with meropenem or colistin against multidrug resistant (MDR) Acinetobacter baumannii isolated from hospitalized patients from a tertiary care hospital in Saudi Arabia. During the study period, 54 multidrug and carbapenem-resistant isolates of A. baumannii isolates were collected from blood and respiratory samples of patients with ventilator-associated pneumonia or bacteremia. Microbroth checkerboard assay (CBA) and E-test were performed to look for synergistic interface of sulbactam and tazobactam with meropenem or colistin. All 54 MDR isolates of A. baumannii were resistant to carbapenem. Minimum inhibitory concentration [50/90] value against sulbactam, tazobactam, meropenem, colistin was found to be 64/128, 64/128, 64/256, and 0.5/1.0 respectively. Synergy was detected in more isolates with CBA compared to E-test. All four combinations showed significant synergistic bactericidal activity. However, the combination with colistin showed greater synergistic effect than combination with meropenem. Antagonism was not detected with any of the combinations and any method, but indifference was seen in tazobactam and colistin combination alone. A significant bactericidal effect was seen with sulbactam combination with meropenem or colistin in both methods. A combination therapy can be a choice of treatment. As colistin is known to exhibit nephrotoxicity, the combination of sulbactam and meropenem might be considered as an alternative antibiotic treatment for such multi- and extremely resistant bacteria. Yet, sample size is small in our study, so further well-designed in vitro and clinical studies on large scale should confirm our findings.

Historical development of typing methods for Streptococcus pneumoniae

Streptococcus pneumoniae is a major cause of morbidity and mortality in developing countries, especially in children, even though this is a vaccine-preventable disease. Several vaccines are available for which programmes are in regular immunization, but the success of this depends upon their efficacy and vaccine coverage of the serotypes prevalent in the region. A so-called replacement phenomenon has made it mandatory that there should be a regular and continuous survey of serotype prevalence whenever the vaccine is used. Many typing methods have been described including serology as well as molecular-based typing. We have analysed most of the described typing methods for pneumococcal isolates and discussed their prospects for future surveillance programmes. In brief, molecular typing such as sequential multiplex PCR is more reliable for typing, but it has limitations as it types the isolates only up to serogroup level in few cases, and therefore is still dependent on conventional serotyping methods like Quellung and co-agglutination. However, conventional typing methods are limited because of cross-reactivity with other serotypes within serogroups. Conventional serological methods are costly, labour-intensive, and prone to misidentification, whereas current DNA-based methods have limited serotype coverage requiring multiple PCR primers. Implementation of pneumococcal conjugate vaccines necessitates continued monitoring of circulating strains to consider vaccine effectiveness and subsequent substitution of serotypes.