Characterization of aminoglycoside resistance genes in multidrug-resistant Klebsiella pneumoniae collected from tertiary hospitals during the COVID-19 pandemic.

Since the peak of the coronavirus disease 2019 (COVID-19) pandemic, concerns around multidrug-resistant (MDR) bacterial pathogens have increased. This study aimed to characterize aminoglycoside resistance genes in MDR Klebsiella pneumoniae (K. pneumoniae) collected during the COVID-19 pandemic. A total of 220 clinical isolates of gram-negative bacteria were collected from tertiary hospitals in Makkah, Saudi Arabia, between April 2020 and January 2021. The prevalence of K. pneumoniae was 40.5%; of the 89 K. pneumoniae isolates, MDR patterns were found among 51 (57.3%) strains. The MDR isolates showed elevated resistance rates to aminoglycoside agents, including amikacin (100%), gentamicin (98%), and tobramycin (98%). PCR assays detected one or more aminoglycoside genes in 42 (82.3%) MDR K. pneumoniae strains. The rmtD gene was the most predominant gene (66.7%; 34/51), followed by aac(6')-Ib and aph(3')-Ia (45.1%; 23/51). The aac(3)-II gene was the least frequent gene (7.8%; 4/51) produced by our isolates. The rmtC gene was not detected in the studied isolates. Our findings indicated a high risk of MDR bacterial infections through the COVID-19 outbreak. Therefore, there is a need for continuous implementation of effective infection prevention control (IPC) measures to monitor the occurrence of MDR pathogens and the emergence of MDR bacterial infections through the COVID-19 outbreak.

The Influence of Different Plasma Cell Discharges on the Performance Quality of Surgical Gown Samples.

An experimental study was performed on a low-density plasma discharge using two different configurations of the plasma cell cathode, namely, the one mesh system electrodes (OMSE) and the one mesh and three system electrodes (OMTSE), to determine the electrical characteristics of the plasma such as current-voltage characteristics, breakdown voltage (VB), Paschen curves, current density (J), cathode fall thickness (dc), and electron density of the treated sample. The influence of the electrical characteristics of the plasma fluid in the cathode fall region for different cathode configuration cells (OMSE and OMTSE) on the performance quality of a surgical gown was studied to determine surface modification, treatment efficiency, exposure time, wettability property, and mechanical properties. Over a very short exposure time, the treatment efficiency for the surgical gown surface of plasma over the mesh cathode at a distance equivalent to the cathode fall distance dc values of the OMTSE and for OMSE reached a maximum. The wettability property decreased from 90 to 40% for OMTSE over a 180 s exposure time and decreased from 90 to 10% for OMSE over a 160 s exposure time. The mechanisms of each stage of surgical gown treatment by plasma are described. In this study, the mechanical properties of the untreated and treated surgical gown samples such as the tensile strength and elongation percentage, ultimate tensile strength, yield strength, strain hardening, resilience, toughness, and fracture (breaking) point were studied. Plasma had a more positive effect on the mechanical properties of the OMSE reactor than those of the OMTSE reactor.

Increasing frequency of Aminoglycoside-Resistant Klebsiella pneumoniae during the era of pandemic COVID-19.

The emergence of multidrug resistance to aminoglycosides in K. pneumoniae isolates is a growing concern, especially during pandemic Coronavirus disease 2019 (COVID-19). The study identifies antibiotic resistance in K. pneumoniae isolated from tertiary hospitals during pandemic COVID-19. Among 220 clinical isolates, the total rate of K. pneumoniae was found to be 89 (40.5%). Phenotyping results confirmed the resistance of aminoglycoside antibiotics in 51 (23.2%) of K. pneumoniae isolates. PCR results confirmed the existence of one or more aminoglycoside genes in 82.3% of the 51 isolates. The rmtD gene was the highest-detected gene (66.7%), followed by aac(6')-Ib (45.1%), aph(3')-Ia (45.1%), rmtB (29.4%), armA (21.6%), aac(3)-II (7.8%), and rmtA (3) (11.8%). Significantly, higher resistance strains showed a higher prevalence (61.5%) of aminoglycoside genes (p < 0.05). During COVID-19, there is a higher risk of acquiring MDR bacterial infections, so the monitoring of multidrug resistant bacteria must be continuously undertaken to implement effective measures in infection control and prevention.

Inactivation of E. coli Using Atmospheric Pressure Plasma Jet with Dry and Wet Argon Discharges.

The acceleration of inactivating viable cells of Escherichia coli (E. coli), by using new direct and indirect innovative methods, is the targeted method of using an atmospheric pressure plasma jet (APPJ) operated by an AC high-voltage power source with variable frequency up to 60 kHz and voltage ranging from 2.5 to 25 kV. Discharges using dry argon (0% O2) discharges and different wet argon discharges using admixtures with O2/Ar ratios ranging from 0.25% to 1.5% were studied. The combined effects of dry and wet argon discharges, direct and indirect exposure using a mesh controller, and hollow magnets were studied to reach a complete bacterial inactivation in short application times. Survival curves showed that the inactivation rate increased as the wettability increased. The application of magnetized non-thermal plasma discharge with a 1.5% wetness ratio causes a fast inactivation rate of microbes on surfaces, and a dramatic decrease of the residual survival of the bacterial ratio due to an increase in the jet width and the enhanced ability of fast transport of the charges to viable cells, especially at the edge of the Petri dish. The membrane damage of E. coli mechanism factors in the activation process by APPJ is discussed.

An efficient system for the generation of marked genetic mutants in members of the genus Burkholderia.

To elucidate the function of a gene in bacteria it is vital that targeted gene inactivation (allelic replacement) can be achieved. Allelic replacement is often carried out by disruption of the gene of interest by insertion of an antibiotic-resistance marker followed by subsequent transfer of the mutant allele to the genome of the host organism in place of the wild-type gene. However, due to their intrinsic resistance to many antibiotics only selected antibiotic-resistance markers can be used in members of the genus Burkholderia, including the Burkholderia cepacia complex (Bcc). Here we describe the construction of improved antibiotic-resistance cassettes that specify resistance to kanamycin, chloramphenicol or trimethoprim effectively in the Bcc and related species. These were then used in combination with and/or to construct a series enhanced suicide vectors, pSHAFT2, pSHAFT3 and pSHAFT-GFP to facilitate effective allelic replacement in the Bcc. Validation of these improved suicide vectors was demonstrated by the genetic inactivation of selected genes in the Bcc species Burkholderia cenocepacia and B. lata, and in the non-Bcc species, B. thailandensis.

Molecular characterization of methicillin-resistant Staphylococcus aureus isolated from tertiary care hospitals.

BACKGROUND AND OBJECTIVES: Methicillin-resistant S. aureus (MRSA) tends to be resistant to multiple antibiotics. Methicillin resistance is conferred by the acquisition of the mecA gene, which is carried by a mobile genetic element called the staphylococcal cassette chromosome mec (SCCmec). There are five major types of SCCmec elements (I-V). The majority of hospital-acquired MRSA (HA-MRSA) strains carry SCCmec types I, II, or III, whereas community-acquired MRSA (CA-MRSA) strains carry SCCmec types IV or V. In addition, Panton-Valentine Leucocidin (PVL) is a gene encoding a powerful cytotoxin that is strongly associated with CA-MRSA strains. The present study was aimed to identify the types of SCCmec and PVL genes among clinical MRSA isolates. METHODS: This study was conducted in 5 tertiary care hospitals in Makkah city from March to September of 2012. A total of 206 S. aureus clinical isolates were analysed using standard microbiological methods. Multiplex PCR was performed on genomic DNA from MRSA isolates in order to identify the types of SCCmec. In addition, PCR was performed to detect the PVL gene among the isolates. RESULTS: Of the 206 S. aureus isolates, 114 (55.3%) were MRSA, and 100 of the MRSA isolates carried the mecA gene. RESULTS from SCCmec typing revealed that 3% were type I; 9% were type II; 47% were type III, and 29% were type IV. Nineteen per cent of the isolates harboured the PVL gene. Furthermore, there was a statistically significant correlation between the presence of the PVL gene and SCCmec type IV. CONCLUSION: The virulence of MRSA strains is increasing in both hospital and community settings in Makkah, highlighting the importance of their rapid identification in order to appropriately control infection.

Frequency and antibiotic susceptibility of gram-positive bacteria in Makkah hospitals.

BACKGROUND AND OBJECTIVE: Gram-positive bacteria are important nosocomial pathogens. The objective of this study was to estimate the frequencies and resistance rates of gram-positive pathogens isolated from hospitals in Makkah, Saudi Arabia. DESIGN AND SETTING: Prospective study at three Makkah hospitals from May 2008 to April 2009. PATIENTS AND METHODS: Clinical isolates were collected and demographic and laboratory data were recorded. Standard microbiological methods were used to identify the organisms and test for antimicrobial susceptibility. The results were interpreted according to the Clinical Laboratory Standards Institute (CLSI) guidelines. RESULTS: Clinical isolates were collected from 1087 patients. Gram-positive pathogens infected all age groups, but had no gender predominance. Staphylococcus aureus was the most common cause of wound infection and accounted for more than half of the clinical isolates (688 cases). Coagulase-negative staphylococcus (CONS) was a common isolate from blood cultures. Wounds were the most common site of infection (37.6%). Enterococcus spp. and Streptococcus agalactiae were the second most common bacteria (26%). The resistance rates of S aureus and CONS isolates were 39.4% and 82.4% for oxacillin, respectively. Among the streptococci, the resistance rates of Streptococcus pneumoniae were 21.1% and 16.7% for ampicillin and erythromycin, respectively. CONCLUSIONS: S aureus infections were found to be very common in the Makkah hospitals. Infection prevention, control measures and continuous monitoring for antibiotic susceptibility are necessary to reduce these and other nosocomial infections.

Profile of bacterial pneumonia during Hajj.

BACKGROUND & OBJECTIVES: The congregation of a large number of people during Hajj seasons from different parts of the world in overcrowded conditions within a confined area for a long period of time presents many public health challenges and health risks. One of the main health problems of the crowding is ease transmission of pneumonia by air droplets. This study was aimed to determine the most common causes of bacterial pneumonia during the 2005 Hajj season and to relate the findings with clinical conditions. METHODS: A total of 141 patients with suspected pneumonia from the three main tertiary care hospitals in Makkah, Saudi Arabia, were investigated during Hajj season, 2005. Sputum and serum samples were collected and investigated for the possible presence of typical or atypical causative agents. RESULTS: Of the 141 clinically suspected pneumonia cases, 76 (53.9%) were confirmed positive by microbiological tests. More than 94 per cent of the confirmed cases were in the age group >50 yr, and 56.6 per cent of the cases were men. The most frequent isolates were Candida albicans (28.7%) and Pseudomonas aeruginosa (21.8%), followed by Legionella pneumophila (14.9%) and Klabsiella pneumoniae (9.2%). More than one causative pathogens were isolated in 15 patients (16.3%), and 55 per cent of patients were diabetic. INTERPRETATION & CONCLUSIONS: Clinicians should be aware that typical pneumonia treatment regimens may not work well during the Hajj season due to the wide variety of isolated organisms. This necessitates taking a sputum sample before starting treatment for identification and sensitivity testing. Special precautions need to be taken for >50 yr old patients.

The pobA gene of Burkholderia cenocepacia encodes a group I Sfp-type phosphopantetheinyltransferase required for biosynthesis of the siderophores ornibactin and pyochelin.

The opportunistic pathogen Burkholderia cenocepacia produces the siderophores ornibactin and pyochelin under iron-restricted conditions. Biosynthesis of both siderophores requires the involvement of non-ribosomal peptide synthetases (NRPSs). Using a transposon containing the lacZ reporter gene, two B. cenocepacia mutants were isolated which were deficient in siderophore production. Mutant IW10 was shown to produce normal amounts of ornibactin but only trace amounts of pyochelin, whereas synthesis of both siderophores was abolished in AHA27. Growth of AHA27, but not IW10, was inhibited under iron-restricted conditions. In both mutants, the transposon had integrated into the pobA gene, which encodes a polypeptide exhibiting similarity to the Sfp-type phosphopantetheinyltransferases (PPTases). These enzymes are responsible for activation of NRPSs by the covalent attachment of the 4'-phosphopantetheine (P-pant) moiety of coenzyme A. Previously characterized PPTase genes from other bacteria were shown to efficiently complement both mutants for siderophore production when provided in trans. The B. cenocepacia pobA gene was also able to efficiently complement an Escherichia coli entD mutant for production of the siderophore enterobactin. Using mutant IW10, in which the lacZ gene carried by the transposon is inserted in the same orientation as pobA, it was shown that pobA is not appreciably iron-regulated. Finally, we confirmed that Sfp-type bacterial PPTases can be subdivided into two distinct groups, and we present the amino acid signature sequences which characterize each of these groups.

Frequency and antimicrobial susceptibility of gram-negative bacteria isolated from 2 hospitals in Makkah, Saudi Arabia.

OBJECTIVE: To estimate the prevalence and antibiotic susceptibility of the gram-negative bacteria isolated from 2 hospitals in Makkah. METHODS: This study was undertaken in 2 main tertiary care hospitals namely; Al-Noor Specialist Hospital, and Hera Hospital in Makkah, Kingdom of Saudi Arabia from October 2005 to March 2006. A total of 1137 gram-negative bacteria were identified in non-duplicate clinical specimens obtained from 965 patients of various body sites infections. Demographic data, identity of microorganisms, and antimicrobial susceptibilities were obtained from medical and laboratory records. RESULTS: The most prevalent gram-negative bacteria were Escherichia coli (31.6%), and Pseudomonas aeruginosa (31.2%), followed by Acinetobacter baumannii (10.8%), Klebsiella pneumoniae (8.3%), Klebsiella sp. (6.2%), Haemophilus influenzae (3.7%), Proteus sp. (3.3%), and Enterobacter sp. (1.9%). Results demonstrated that gram-negative bacteria have a high rate of resistance to commonly used antibiotics. Furthermore, multi-drug resistance was also common in this study. CONCLUSION: Our data showed a high rate of resistance among gram-negative pathogens in comparison with other countries in the world. The implementation of monitoring programs is an important part of the prevention strategy against the development of antibiotic resistance in hospitals.

Frequency and antimicrobial susceptibility patterns of bacterial pathogens isolated from septicemic patients in Makkah hospitals.

OBJECTIVES: To determine the frequency of septicemic cases in Makkah hospitals, the main pathogens causing septicemia, and to describe the prevalence of antibiotic resistance among septicemia clinical isolates. METHODS: We performed a prospective study of 1626 septicemic cases in the 4 main hospitals in Makkah City during April 2004 to March 2005. Blood culture, isolation of organism and susceptibility to antibiotics were assessed using a routine microbiological methods. RESULTS: Out of the 1626 septicemic cases identified, gram-positive organisms were involved in 56.6% of these episodes with coagulase-negative Staphylococci and Staphylococcus aureus being the most frequent. While Escherichia coli and Pseudomonas species were the most common among gram-negative organisms. Candida species was involved in 5.9% of all encountered organisms. Most septicemic cases were reported in male patients over 50 years, the intensive care units, Saudi patients and during Hajj season. Results also showed the frequency and antimicrobial susceptibility patterns of bacterial pathogens isolated from septicemic patients in Makkah hospitals. CONCLUSION: The rates of antibiotic resistance among pathogens in this study, are much higher than what has been reported elsewhere in the Kingdom as well as in many of international studies.

Methicillin resistance among Staphylococcus aureus isolates from Saudi hospitals.

OBJECTIVE: To determine the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) strains among clinical isolates collected from the 4 tertiary hospitals in Makkah, Saudi Arabia, and to test the antimicrobial susceptibility patterns of S. aureus isolates against 9 antimicrobial agents. MATERIALS AND METHODS: A total of 512 S. aureus clinical isolates were collected during a period of 1 year starting in April 2003 in Al-Noor, King Abdul-Aziz, Hera and King Faisal Hospitals, Makkah, Saudi Arabia. The sensitivity patterns of these isolates were determined using the Kirby-Bauer disk diffusion method. RESULTS: The prevalence of MRSA among S. aureus isolates was 38.9% (199/512). Among 199 MRSA isolates, 78.8% showed multidrug resistance to erythromycin, gentamicin and oxytetracycline. CONCLUSION: The rate of MRSA resistance in this study was much higher than what had been reported in other areas of Saudi Arabia emphasizing the need for local or country-based surveillance to characterize and monitor MRSA and to develop strategies that will improve MRSA treatment and control.

Accuracy of current oxacillin sensitivity tests routinely used in hospitals in Western Saudi Arabia.

OBJECTIVE: To determine the accuracy of the current oxacillin resistant Staphylococcus aureus (S. aureus) detection test used in Makkah hospitals in comparison with the National Committee for Clinical Laboratory Standards (NCCLS) method. METHODS: A total of 500 S. aureus clinical isolates and it's oxacillin sensitivity patterns were collected from the 4 main hospitals in Makkah, Kingdom of Saudi Arabia between April 2003 and January 2004. The oxacillin sensitivity of these clinical isolates were re-examined using the NCCLS standard method and confirmed using polymerase chain reaction (PCR) technique. RESULTS: Of 500 clinical isolates, 103 (20.6%) were resistant to oxacillin using NCCLS standard method but they were sensitive according to the current hospital routine sensitivity test method. The PCR technique confirmed the presence of mecA gene in 88/103 isolates appeared to be methicillin-resistant S. aureus (MRSA) using NCCLS standard technique. CONCLUSION: A significant percentage of MRSA are currently misdiagnosed in accordance with the current routine sensitivity method. In addition, some mecA negative and oxacillin resistant strains (according to the NCCLS standard method) can be misdiagnosed by using PCR technique. These findings emphasis the urgent need to comply with the recommended NCCLS guidelines for detection of oxacillin resistance. Moreover, the PCR technique can not be used as a single diagnostic tool for detection of MRSA.

The Burkholderia cepacia fur gene: co-localization with omlA and absence of regulation by iron.

The ferric uptake regulator (Fur) functions as a transcription repressor of many genes in bacteria in response to iron, but the presence of a functional equivalent of this protein has not been demonstrated in Burkholderia cepacia. A segment of the Burkholderia pseudomallei fur gene was amplified using degenerate primers and used to identify chromosomal restriction fragments containing the entire fur genes of B. cepacia and B. pseudomallei. These fragments were cloned and sequenced, revealing the Fur protein of both species to be a polypeptide of 142 amino acids possessing a high degree of amino acid sequence identity to Fur of other members of the beta subclass of the Proteobacteria. Primer extension analysis demonstrated that transcription of B. cepacia fur originated from a single promoter located 36 bp upstream from the fur translation initiation codon. The Fur polypeptide of B. cepacia was shown to functionally substitute for Fur in an Escherichia coli fur mutant. Single copy fur-lacZ fusions were constructed and used to examine the regulation of B. cepacia fur. The B. cepacia fur promoter was not responsive to iron availability, the presence of hydrogen peroxide or the superoxide generator methyl viologen. In addition, fur expression was not significantly influenced by carbon source. Interestingly, the presence of the divergently transcribed omlA/smpA gene upstream of fur in some members of the gamma subclass of the Proteobacteria is retained in several genera within the beta taxon, including Burkholderia.