Bioactive 2-(Methyldithio)Pyridine-3-Carbonitrile from Persian Shallot (Allium stipitatum Regel.) Exerts Broad-Spectrum Antimicrobial Activity.

Antibiotic resistance is a problem that continues to challenge the healthcare sector, especially in clinically significant pathogens like methicillin-resistant Staphylococcus aureus (MRSA). Herein is described the isolation and structure elucidation of a bioactive compound from Allium stipitatum with antimicrobial activity. Crude Allium stipitatum dichloromethane extract (ASDE) was subjected to systematic purification by chromatographic procedures to afford various bioactive fractions. A fraction that exhibited anti-MRSA activity (4 µg·mL-1) was further characterized to determine the structure. The structure of the compound was elucidated as 2-(methyldithio)pyridine-3-carbonitrile (2-Medpy-3-CN). The 2-Medpy-3-CN compound, which was screened for antimicrobial activity, exhibited minimum inhibitory concentrations (MICs) in the range of 0.5 to >64 µg·mL-1 for tested bacterial species and 0.25 to 2 µg·mL-1 for Candida spp. Further studies are important to confirm the drug target and mechanism of action.

The mazEF toxin-antitoxin system as a novel antibacterial target in Acinetobacter baumannii.

Although analysis of toxin-antitoxin (TA) systems can be instructive, to date, there is no information on the prevalence and identity of TA systems based on a large panel of Acinetobacter baumannii clinical isolates. The aim of the current study was to screen for functional TA systems among clinical isolates of A. baumannii and to identify the systems' locations. For this purpose, we screened 85 A. baumannii isolates collected from different clinical sources for the presence of the mazEF, relBE and higBA TA genes. The results revealed that the genes coding for the mazEF TA system were commonly present in all clinical isolates of A. baumannii. Reverse transcriptase-polymerase chain reaction analysis showed that transcripts were produced in the clinical isolates. Our findings showed that TA genes are prevalent, harboured by chromosomes and transcribed within A. baumannii. Hence, activation of the toxin proteins in the mazEF TA system should be investigated further as an effective antibacterial strategy against this bacterium.

Wide distribution of virulence genes among Enterococcus faecium and Enterococcus faecalis clinical isolates.

Enterococcus, a Gram-positive facultative anaerobic cocci belonging to the lactic acid bacteria of the phylum Firmicutes, is known to be able to resist a wide range of hostile conditions such as different pH levels, high concentration of NaCl (6.5%), and the extended temperatures between 5(°)C and 65(°)C. Despite being the third most common nosocomial pathogen, our understanding on its virulence factors is still poorly understood. The current study was aimed to determine the prevalence of different virulence genes in Enterococcus faecalis and Enterococcus faecium. For this purpose, 79 clinical isolates of Malaysian enterococci were evaluated for the presence of virulence genes. pilB, fms8, efaAfm, and sgrA genes are prevalent in all clinical isolates. In conclusion, the pathogenicity of E. faecalis and E. faecium could be associated with different virulence factors and these genes are widely distributed among the enterococcal species.

Modified silver staining in 2DE improves protein detection even at extremely low sample concentration.

The typical concentration of protein loaded varies from 0.13 to 1.40 µg/µL for a classical silver staining method in 2DE gel. Here, we present a simple modified classical silver staining method by modifying the silver impregnation and development reaction steps. This modified method detects the protein spots at extremely low loaded concentrations, ranging from 0.0048 to 0.0480 µg/µL. We recommend this modified silver staining as an excellent method for the limited biological samples used for silver-stained 2DE analysis. Altogether, the protocol takes close to two days from first dimension separation to second dimension separation, followed by silver staining, scanning, and analysis.

Comparative Exoproteome Profile of MRSA-ST9 Isolated from Pigs and Pig Handler.

MRSA-ST9-t4358 from pigs and pig handlers were analysed for exoproteins profiles. Similar protein patterns with variation in protein spot intensity were observed. No significant variation among the protein profiles indicates that transmission of MRSA-ST9 from pig to human or vice versa, do not strongly alter the gene expression patterns. Indirectly, virulence factors that acquired from pig environment might have chance to be expressed in human host.

Methicillin-susceptible Staphylococcus aureus from clinical and community sources are genetically diverse.

Despite the association of methicillin-susceptible S. aureus (MSSA) with several life-threatening diseases, relatively little is known about their clinical epidemiology in Malaysia. We characterized MSSA isolates (n=252) obtained from clinical and community (carriage) sources based on spa sequencing and multilocus sequence typing (MLST). The prevalence of several important virulence genes was determined to further define the molecular characteristics of MSSA clones circulating in Malaysia. Among the 142 clinical and 110 community-acquired MSSA isolates, 98 different spa types were identified, corresponding to 8 different spa clonal clusters (spa-CCs). In addition, MLST analysis revealed 22 sequence types (STs) with 5 singletons corresponding to 12 MLST-CCs. Interestingly, spa-CC084/085 (MLST-CC15) (p=0.038), spa-non-founder 2 (MLST-ST188) (p=0.002), and spa-CC127 (MLST-CC1) (p=0.049) were identified significantly more often among clinical isolates. spa-CC3204 (MLST-CC121) (p=0.02) and spa-CC015 (MLST-CC45) (p=0.0002) were more common among community isolates. Five dominant MLST-CCs (CC8, CC121, CC1, CC45, and CC5) having clear counterparts among the major MRSA clones were also identified in this study. While the MSSA strains are usually genetically heterogeneous, a relatively high frequency (19/7.5%) of ST188 (t189) strains was found, with 57.8% of these strains carrying the Panton-Valentine leukocidin (PVL). Analysis of additional virulence genes showed a frequency of 36.5% and 36.9% for seg and sei and 0.8% and 6.3% for etb and tst genes, respectively. Arginine catabolic mobile element (ACME) was detected in 4 community isolates only. These represent the first isolates harbouring this gene in an Asian region. In conclusion, MSSA from the Malaysian community and their clinical counterparts are genetically diverse, but certain clones occur more often among clinical isolates than among carriage isolates and vice versa.

Predominance and emergence of clones of hospital-acquired methicillin-resistant Staphylococcus aureus in Malaysia.

We define the epidemiology of predominant and sporadic methicillin-resistant Staphylococcus aureus (MRSA) strains in a central teaching and referral hospital in Kuala Lumpur, Malaysia. This is done on the basis of spa sequencing, multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec) typing, and virulence gene profiling. During the period of study, the MRSA prevalence was 44.1%, and 389 MRSA strains were included. The prevalence of MRSA was found to be significantly higher in the patients of Indian ethnicity (P < 0.001). The majority (92.5%) of the isolates belonged to ST-239, spa type t037, and possessed the type III or IIIA SCCmec. The arginine catabolic mobile element (ACME) arcA gene was detected in three (1.05%) ST-239 isolates. We report the first identification of ACME arcA gene-positive ST-239. Apart from this predominant clone, six (1.5%) isolates of ST-22, with two related spa types (t032 and t4184) and a singleton (t3213), carrying type IVh SCCmec, were detected for the first time in Asia. A limited number of community-acquired (CA) MRSA strains were also detected. These included ST-188/t189 (2.1%), ST-1/t127 (2.3%), and ST-7/t091 (1%). Panton-Valentin leukocidin (PVL) was detected in all ST-1 and ST-188 strains and in 0.7% of the ST-239 isolates. The majority of the isolates carried agr I, except that ST-1 strains were agr III positive. Virulence genes seg and sei were seen only among ST-22 isolates. In conclusion, current results revealed the predominance of ST-239-SCCmec III/IIIA and the penetration of ST-22 with different virulence gene profiles. The emergence in Malaysia of novel clones of known epidemic and pathogenic potential should be taken seriously.

Prevalence of ST9 methicillin-resistant Staphylococcus aureus among pigs and pig handlers in Malaysia.

Methicillin-resistant Staphylococcus aureus (MRSA) of sequence type 398 (ST398) has frequently been detected in pigs and pig handlers. However, in Malaysia, sampling 360 pigs and 90 pig handlers from 30 farms identified novel ST9-spa type t4358-staphylococcal cassette chromosome mec type V MRSA strains that were found to transiently colonize more than 1% of pigs and 5.5% of pig handlers.

Microhabitat Factors Influenced the Prevalence of Pathogenic Leptospira spp. in Small Mammal Host.

Leptospirosis, a widespread zoonotic disease, is a public health problem, especially in major urban centres, and is mainly reported to be associated with rats. In Malaysia, focus has been primarily given to the Leptospira prevalence in rodents per se, but there is lack of information on the microhabitat structure of the outbreak areas. We aimed to determine the diversity of small mammal species, microhabitat types, and their prevalence of pathogenic Leptospira spp. in the outbreak areas, which were categorized as urban, semi-urban, and recreational forests. Sampling involved deploying 100 to 300 live traps at each study site. Kidney samples were extracted from selected individuals, for screening of pathogenic Leptospira spp. by PCR. Out of 537 individuals from 15 small mammal species captured, 4 species were recorded from urban, 13 from semi-urban, and 11 from recreational forest sites. From 389 individuals screened, 58 were tested positive for pathogenic Leptospira. Recreational forests recorded the highest prevalence with 19.4% (n = 93), followed by urban, 16.6% (n = 163) and semi-urban sites with 9.8% (n = 133). Seven rodent species were tested positive for pathogenic Leptospira from all areas. R. norvegicus was found to harbour the highest prevalence (66.7%) in urban, R. rattus (53.8%) in semi-urban, whereby M. whiteheadi (44.4%) in recreational forest sites. Microhabitat analysis revealed that rubbish quantity contributed especially strongly to a high prevalence of Leptospira. This study contributes to understanding of the host and microhabitat preferences of Leptospira, which is important in controlling the spread of this disease in human's landscapes.

Antibacterial and Antibiofilm Activities of Nonpolar Extracts of Allium stipitatum Regel. against Multidrug Resistant Bacteria.

The present study assessed the in vitro antibacterial and antibiofilm potential of hexane (ASHE) and dichloromethane (ASDE) extracts of Allium stipitatum (Persian shallot) against planktonic cells and biofilm structures of clinically significant antibiotic resistant pathogens, with a special emphasis on methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA), and emerging pathogens, Acinetobacter baumannii and Stenotrophomonas maltophilia. Antibacterial activities were determined through disk diffusion, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), time-kill kinetics, and electron microscopy. Antibiofilm activity was assessed by XTT [2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide] reduction assay and by confocal laser scanning microscopy (CLSM). The zone of inhibition ranged from 13 to 33 mm, while the MICs and MBCs ranged from 16 to 1024 µg mL-1. Both ASHE and ASDE completely eradicated overnight cultures of the test microorganisms, including antibiotic resistant strains. Time-kill studies showed that the extracts were strongly bactericidal against planktonic cultures of S. aureus, MRSA, Acinetobacter baumannii, and S. maltophilia as early as 4 hours postinoculation (hpi). ASHE and ASDE were shown to inhibit preformed biofilms of the four biofilm phenotypes tested. Our results demonstrate the potential therapeutic application of ASHE and ASDE to inhibit the growth of gram-positive and gram-negative biofilms of clinical significance and warrant further investigation of the potential of A. stipitatum bulbs against biofilm-related drug resistance.

Prevalence and molecular characterisation of Schistosoma haematobium among primary school children in Kebbi State, Nigeria

Schistosomiasis is the major source of morbidity in Sub-Saharan Africa and Asia. It is estimated that 207 million people are infected, of which 97% are in Africa. The aim of this study was the determining of prevalence as well as the phylogeny of S. haematobium among school children in Argungu Emirate, Kebbi State Nigeria. A total of 325 urine samples was collected from school children between 7 to 14 years. S. heamatobium eggs was examined under dissecting microscope and DNA was extracted from urine sample and COX1 gene was amplified by nested PCR. The PCR products were purified, sequenced and analysed. This study showed a prevalence of 32.09%, with male pupils having the highest prevalence. S. haematobium infections in children who fetch water in the river have 24 times higher risk of being infected while those who bath in the river have 158 times higher risk of being infected. Our sequences were phylogenetically related to S. haematobium isolate U82266 from Kenya and consistence with the predominant species in Africa. This was the first S. haematobium and S. mansoni co-infection reported in Nigeria. S. haematobium infection is prevalent among school age and significantly associated with water contact.

Allium stipitatum Extract Exhibits In Vivo Antibacterial Activity against Methicillin-Resistant Staphylococcus aureus and Accelerates Burn Wound Healing in a Full-Thickness Murine Burn Model.

The in vivo antibacterial and burn wound healing potency of Persian shallot bulbs (Allium stipitatum) were explored in a mice burn model infected with methicillin-resistant Staphylococcus aureus (MRSA). Hexane (ASHE) and dichloromethane (ASDE) extracts were tested. Female BALB/c mice were inflicted with third-degree thermal injury followed by infection with MRSA. ASHE and ASDE formulated with simple ointment base (SOB) at concentrations of 1%, 2%, and 5% (w/w) were topically applied to burn wounds twice a day for 20 days. Silver sulfadiazine (1%) served as drug positive control. Microbiological analysis was carried out on 1, 2, 3, 4, and 5 days postwounding (dpw) and histopathological analysis at the end of the experiment (20 dpw). Both ointments demonstrated strong antibacterial activity with complete elimination of MRSA at 48-72 h after infection. The rate of wound contraction was higher (95-100%) in mice groups treated with ASHE and ASDE ointments after 15 dpw. Histological analysis revealed significant increase (p < 0.05) in epithelialization and collagenation in treated groups. The ASHE and ASDE were found to be relatively noncytotoxic and safe to Vero cell line (383.4 µg mL-1; 390.6 µg mL-1), suggesting the extracts as safe topical antibacterial as well as promising alternatives in managing thermal injuries.

A Low Prevalence of Inducible Macrolide, Lincosamide, and Streptogramin B Resistance Phenotype among Methicillin-Susceptible Staphylococcus aureus Isolated from Malaysian Patients and Healthy Individuals.

BACKGROUND: Antibiotic resistance among Staphylococcus aureus is of great concern worldwide. This resistance is further complicated by the ability of S. aureus to confer cross-resistance to other antibiotics due to the presence of resistance genes, such as erythromycin resistance methylase (erm) genes, which render the bacterium resistant to macrolide-lincosamide-streptogramin B (MLSB) antibiotics. Resistance to these antibiotics can lead to therapeutic failure, resulting in significant morbidity and mortality in patients with S. aureus infections. OBJECTIVES: This study was performed to examine the distribution of MLSB-resistant strains of methicillin-susceptible S. aureus (MSSA), which were obtained from hospitalized patients and normal healthy individuals (carriers) using phenotypic methods, such as the double-disk diffusion (D-test) and the genotypic method by polymerase chain reaction (PCR). METHODS: A total of 183 nonduplicative MSSA isolates obtained from hospitalized patients (133) and carriers (50) in our previous studies were randomly selected for the D-test. The guidelines of the Clinical and Laboratory Standards Institute (CLSI) were used for the interpretation of the results of this test. The detection of ermA, ermB, ermC and msrA genes by PCR was performed for isolates that had positive D-test results and that were resistant to erythromycin. RESULTS: Of the 183 MSSA isolates, 97.2% and 98.4% were highly susceptible to erythromycin and clindamycin, respectively. MSLB resistance was detected in four isolates (2.2%). Of the 133 MSSA isolated from hospitalized patients, only 3.0% (4/133) and 2.3% (3/133) exhibited resistance to erythromycin and clindamycin, respectively. With regard to the MLSB resistance phenotypes, only 1.6% and 0.6% exhibited inducible MLSB (iMLSB) and MS phenotypes, respectively. The ermC gene was detected in all three iMLSB phenotypes, and the msrA gene was detected in the MS phenotype. Surprisingly, all MSSA isolates (100%) from carriers exhibited extremely high susceptibility to both antibiotics. CONCLUSIONS: The prevalence rates of iMLSB MSSA isolates vary according to geographical locations and the local antibiotic policy. The low prevalence rate of iMLSB MSSA isolates could probably be related to the judicious use of antibiotics for treating S. aureus infections in our studied population. Nonetheless, continuous antibiotic surveillance is still necessary to control any emergence of resistance isolates so that targeted therapy and effective control can be implemented accordingly.

Comparative Exoproteomics and Host Inflammatory Response in Staphylococcus aureus Skin and Soft Tissue Infections, Bacteremia, and Subclinical Colonization.

The exoproteome of Staphylococcus aureus contains enzymes and virulence factors that are important for host adaptation. We investigated the exoprotein profiles and cytokine/chemokine responses obtained in three different S. aureus-host interaction scenarios by using two-dimensional gel electrophoresis (2-DGE) and two-dimensional immunoblotting (2D-IB) combined with tandem mass spectrometry (MS/MS) and cytometric bead array techniques. The scenarios included S. aureus bacteremia, skin and soft tissue infections (SSTIs), and healthy carriage. By the 2-DGE approach, 12 exoproteins (the chaperone protein DnaK, a phosphoglycerate kinase [Pgk], the chaperone GroEL, a multisensor hybrid histidine kinase, a 3-methyl-2-oxobutanoate hydroxymethyltransferase [PanB], cysteine synthase A, an N-acetyltransferase, four isoforms of elongation factor Tu [EF-Tu], and one signature protein spot that could not be reliably identified by MS/MS) were found to be consistently present in more than 50% of the bacteremia isolates, while none of the SSTI or healthy-carrier isolates showed any of these proteins. By the 2D-IB approach, we also identified five antigens (methionine aminopeptidase [MetAPs], exotoxin 15 [Set15], a peptidoglycan hydrolase [LytM], an alkyl hydroperoxide reductase [AhpC], and a haptoglobin-binding heme uptake protein [HarA]) specific for SSTI cases. Cytokine and chemokine production varied during the course of different infection types and carriage. Monokine induced by gamma interferon (MIG) was more highly stimulated in bacteremia patients than in SSTI patients and healthy carriers, especially during the acute phase of infection. MIG could therefore be further explored as a potential biomarker of bacteremia. In conclusion, 12 exoproteins from bacteremia isolates, MIG production, and five antigenic proteins identified during SSTIs should be further investigated for potential use as diagnostic markers.

In vitro antibacterial effects of Cinnamomum extracts on common bacteria found in wound infections with emphasis on methicillin-resistant Staphylococcus aureus.

ETHNOPHARMACOLOGICAL RELEVANCE: Cinnamomum species have been widely used in many traditional systems of medicine around the world. In the Malaysian traditional system of medicine, the leaves, stem bark and stem wood of Cinnamomum iners, Cinnamomum porrectum, Cinnamomum altissimum and Cinnamomum impressicostatum have been used to treat wound infections. To study the antibacterial effects of Cinnamomum iners, Cinnamomum porrectum, Cinnamomum altissimum and Cinnamomum impressicostatum against common bacteria found in wound infections with primary focus on methicillin-resistant Staphylococcus aureus (MRSA). MATERIALS AND METHODS: The crude extracts from the leaves, stem-bark and stem-wood of Cinnamomum iners, Cinnamomum porrectum, Cinnamomum altissimum and Cinnamomum impressicostatum were obtained using sequential extraction with hexane, ethylacetate, methanol and water. The volatile oils were obtained by hydro-distillation. The antibacterial activities of extracts were investigated using disk diffusion assays and broth microdilution assays. RESULTS: The volatile oils obtained from the stem-bark of Cinnamomum altissimum, Cinnamomum porrectum and Cinnamomum impressicostatum have shown significant antibacterial activity against a wide range of Gram positive and Gram negative bacteria including MRSA. A few test extracts have shown better activity against MRSA as compared to methicillin sensitive Staphylococcus aureus (MSSA). Amongst all the test extracts, Cinnamomum impressicostatum stem-bark water extract produced the largest inhibition zone of 21.0mm against MRSA while its inhibition zone against MSSA was only 8.5mm. The minimum inhibitory concentration (MIC) of this extract against MRSA was 19.5 µg mL(-1) and the corresponding minimum bactericidal concentration (MBC) was 39.0 µg mL(-1). CONCLUSIONS: This study has scientifically validated the traditional use of Cinnamomum species in treating wound infections. Of high scientific interest was the observation that the antibacterial effect of Cinnamomum impressicostatum stem-bark crude water extract against MRSA was significantly higher than its effect against MSSA, suggesting that the extract contains a compound(s) with higher specific neutralising activity against the drug resistance markers of MRSA.

Methicillin-susceptible and -resistant Staphylococcus aureus with high-level antiseptic and low-level mupirocin resistance in Malaysia.

The prevalence and spread of mupirocin and antiseptic resistance among colonizing and infectious Staphylococcus aureus were determined. S. aureus isolated from anterior nares and infection sites of patients hospitalized in the largest tertiary care referral hospital in Malaysia was investigated for mupirocin and antiseptic susceptibility testing, and for PCR detection of mupA, qacA/B, and smr genes. Twelve isolates showed resistance to mupirocin by disk diffusion, of which 10 (3.8%) harbored the mupA gene. Minimum inhibitory concentrations (MICs) ranged from 64 to 768 µg/ml for mupA positive and below 46 µg/ml for negative isolates. The mupA was more common among ST239 isolates (70%). The qacA/B was carried in 67 out of 95 methicillin-resistant Staphylococcus aureus (MRSA) (70.5%) and 3 out of 164 methicillin-susceptible Staphylococcus aureus (MSSA) (1.8%), while smr was carried in 6 out of 95 MRSA (6.3%) strains. MICs ranged from 3.9 to 15.6 µg/ml for benzethonium chloride (BTC) and benzalkonium chloride (BKC), and from 10.3 to 20.7 µg/ml for chlorhexidine digluconate (CHG). Isolates with qacA/B and smr or qacA/B alone showed higher MIC (20.7 µg/ml for CHG and 15.6 µg/ml for BTC and BKC) than the isolates that lacked antiseptic resistance genes (10.3 µg/ml for CHG and 3.9 µg/ml for BTC and BKC). In 16 cases, ST239 was isolated from the infection site and the nares simultaneously, and shared identical resistance patterns (qacAB or qacAB+smr), suggesting possible endogenous infection. Spread of low-level mupirocin resistance expressing ST239 MRSA and high-level resistance expressing emerging ST1, co-existing with antiseptic-resistant genes showing elevated MICs, should be monitored for effective infection control.

In vitro antibacterial and antibiofilm activities of chlorogenic acid against clinical isolates of Stenotrophomonas maltophilia including the trimethoprim/sulfamethoxazole resistant strain.

The in vitro antibacterial and antibiofilm activity of chlorogenic acid against clinical isolates of Stenotrophomonas maltophilia was investigated through disk diffusion, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), time-kill and biofilm assays. A total of 9 clinical S. maltophilia isolates including one isolate resistant to trimethoprim/sulfamethoxazole (TMP/SMX) were tested. The inhibition zone sizes for the isolates ranged from 17 to 29 mm, while the MIC and MBC values ranged from 8 to 16 µg mL(-1) and 16 to 32 µg mL(-1). Chlorogenic acid appeared to be strongly bactericidal at 4x MIC, with a 2-log reduction in viable bacteria at 10 h. In vitro antibiofilm testing showed a 4-fold reduction in biofilm viability at 4x MIC compared to 1x MIC values (0.085 < 0.397 A 490 nm) of chlorogenic acid. The data from this study support the notion that the chlorogenic acid has promising in vitro antibacterial and antibiofilm activities against S. maltophilia.

Simple, time saving pulsed-field gel electrophoresis protocol for the typing of Stenotrophomonas maltophilia.

We developed a time-saving and cost-efficient Pulsed Field Gel Electrophoresis (PFGE) method for the typing of Stenotrophomonas maltophilia by modifying the conventional procedures. Our modifications related to the cell suspension preparation, lysis of bacterial cells in plugs, washing steps, and consumption of restriction enzyme. Although few rapid PFGE protocols on Gram-negative bacteria are available, the use of comparatively large amounts of costly reagents prompted us to look for other alternative. Hence, by considering the speed, simplicity, and relatively low cost, the modified protocol may be of more practical value than other established protocols in investigating S. maltophilia nosocomial outbreaks.

Short repeats in the spa gene of Staphylococcus aureus are prone to nonsense mutations: stop codons can be found in strains isolated from patients with generalized infection.

Fifteen sequences with stop codons have been obtained in the course of standard methicillin-resistant Staphylococcus aureus (MRSA) spa typing. In nine of those sequences, stop codons occurred due to nonsense G-T and A-T transversions. G-T transversions would appear to be frequent in the spa gene, mostly due to symmetric mutational AT-pressure in the whole S. aureus genome and due to replication-associated mutational pressure characteristic of lagging strands of the "chromosome". A-T transversions would appear to be frequent in the spa gene mostly due to transcription-associated mutational pressure. Relative to other S. aureus genes, short repeats in spa are enriched by nonsense sites for G-T and A-T transversions; the probability of being nonsense for A-T transversion is high in that part of spa coding region. 13 out of 15 (87%) of the sequences with stop codons were obtained from strains isolated from patients with generalized S. aureus infection. Truncation of spa at its C-terminus is predicted to result in a protein that possesses functional IgG binding domains unable to be linked to the cell wall. This is discussed in light of the known fact that extracellular spa is a strong virulence factor involved in immune evasion.

Incidence of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae in patients with urinary tract infection.

CONTEXT AND OBJECTIVES: Resistant bacteria are emerging worldwide as a threat to favorable outcomes from treating common infections in community and hospital settings. The present investigation was carried out to study the incidence of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae in patients with urinary tract infection in different seasons of the year, in order to determine the prevalence of the genes blaTEM, blaSHV and blaCTX-M, which are responsible for ESBL production among ESBL-producing K. pneumoniae, in three cities in Iran, and to investigate the antimicrobial susceptibility pattern of K. pneumoniae in different seasons. DESIGN AND SETTING: Retrospective study carried out among patients with urinary tract infections in five hospitals in Iran. METHOD: Two hundred and eighty-eight clinical isolates of K. pneumoniae were collected between March 2007 and April 2008 from five hospitals in three cities in Iran. ESBLs were identified by phenotypic and genotypic methods. ESBL-producing Klebsiella pneumoniae were evaluated against non-beta-lactam antibiotics. Genes coding for ESBLs (blaSHV, TEM and CTX-M) were screened. RESULTS: Among the 288 clinical isolates of K. pneumoniae, 37.7%, 46.7% and 15.6% were obtained from hospitals in Ilam, Tehran and Tabriz, respectively, of which 39.4%, 50.7% and 45.8% were ESBL-producing K. pneumoniae in Ilam, Milad and Emam Reza hospitals, respectively. CONCLUSION: According to the results from this study, resistance to third-generation cephalosporins is higher during the cold months than during the warm months.