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1.  In Vitro Sensitivity of Salmonella to Ten Antimicrobial Agents Including Sulfamethoxazole and Trimethoprim, Alone and in Combination 
The activities of trimethoprim (TMP) and sulfamethoxazole (SMZ), alone and in combination (SMZ-TMP), and of the following antibiotics were tested against 115 clinical isolates of nontyphoid Salmonella species: tobramycin, gentamicin, ampicillin, amoxicillin, neomycin, kanamycin, chloramphenicol, and tetracycline. The methods of disk diffusion, microtiter broth dilution, and agar dilution were employed for all single antimicrobial agents as well as for SMZ-TMP studies. Growth curves were performed in broth. SMZ-TMP, TMP, gentamicin, tobramycin, and neomycin were the most active drugs in vitro. All strains were inhibited by ≤1 μg of TMP per ml, but >100 μg of SMZ per ml was required for at least 10% of strains. SMZ and TMP in a ratio of 10:0.5, respectively, inhibited all isolates and were synergistic for 105 strains. All strains inhibited by the combination of 10:0.5 SMZ-TMP had a zone diameter of ≥22 mm by using a combination disk containing 1.25 μg of TMP and 23.75 μg of SMZ. Seven isolates were resistant to >100 μg/ml of ampicillin or amoxicillin; all isolates were sensitive to chloramphenicol at ≤6.3 μg/ml. SMZ-TMP appears to be active against nontyphoid salmonellae in vitro; this is usually due to a synergistic effect.
PMCID: PMC444594  PMID: 4791490
2.  Prevalence and susceptibility of uropathogens: a recent report from a teaching hospital in Bangladesh 
BMC Research Notes  2015;8:416.
Background
This investigation was aimed to determine the current status of prevalence and antimicrobial susceptibility of uropathogens isolated in a teaching hospital in Bangladesh. A retrospective analysis was done at the department of Microbiology of Islami Bank Medical College, Rajshahi (IBMCR), Bangladesh during January to December, 2012. Midstream clean-catch urine samples were collected from 443 suspected urinary tract infection patients of different age and sex groups. Uropathogens were identified by standard and specific microbiological techniques and antimicrobial susceptibility pattern was determined by Kirby Bauer Disc diffusion method following Clinical and Laboratory Standards Institute (CLSI) guidelines.
Findings
Culture yielded a total of 189 (42.66 %) significant growths of uropathogens including 179 (94.71 %) unimicrobial (single bacterial species) and 10 (5.29 %) polymicrobial (pair of two different bacterial species) growths. Gender distribution showed 34.44 % male and 48.29 % female UTI patients with male to female ratio of 1:1.46, respectively. E. coli was the predominant isolate (59.30 %), followed by Staph saprophyticus (19.09 %), Enterococcus spp. (11.56 %), Klebsiella spp. (5.53 %), Pseudomonas spp. (2.01 %), Proteus spp. (1.51 %) and Enterobacter spp. (1.00 %). Very high frequency of resistance ranging from 72.03 to 91.53 % to cotrimoxazole, ciprofloxacin, cefuroxime, cephradin, amoxicillin and nalidixic acid, moderately high resistance to ceftriaxone (55.08 %) and gentamicin (40.68 %) and low resistance to nitrofurantoin (16.10 %) were shown by E. coli. Similarly, Staph. saprophyticus and Enterococcus spp. showed low resistance (18.42 and 21.74 %) to nitrofurantoin, but moderately high against cefaclor, gentamycin, cefuroxime and ceftriaxone. Klebsiella spp. and Proteus spp. were 72.73 and 66.67 % susceptible, respectively to gentamycin only but low frequency of susceptibility (<50 %) was found to all other antimicrobial agents. Peudomonas spp. was 75 % susceptible to nitrofurantoin only and showed 75–100 % resistance to all other agents. Enterobacter spp. were 50 % resistant to nitrofurantoin, gentamycin, cefuroxime, cefaclor and ceftriaxone but showed 100 % resistance to all remaining antimicrobials.
Conclusions
Current uropathogens showed the highest rate of susceptibility to nitrofurantoin and gentamicin which can be adapted for empirical treatment of urinary tract infections.
doi:10.1186/s13104-015-1408-1
PMCID: PMC4560919  PMID: 26342570
UTI; Uropathogens; Antibiogram; Teaching hospital; Bangladesh
3.  Antimicrobial Susceptibility of Udder Pathogens Isolated from Dairy Herds in the West Littoral Region of Uruguay 
Acta Veterinaria Scandinavica  2002;43(1):31-41.
A total of 522 strains belonging to streptococci, enterococci and staphylococci isolated from sub-clinical and clinical cases of bovine mastitis from the west littoral region of Uruguay were analysed for their susceptibility to several antimicrobial agents. The susceptibility patterns were studied by agar disk diffusion methods (ADDM) and broth micro-dilution to determine the minimum inhibitory concentration (MIC). The concentration that inhibits 90% (MIC90) of the analysed strains reported in micrograms per millilitre, for Staphylococcus aureus were > 8, 8, ≤ 0.5, ≤ 4, ≤ 1, ≤ 0.5, > 64, ≤ 0.25, 0.5, ≤ 1 and ≤ 1 to penicillin, ampicillin, oxacillin, cephalotin, gentamicin, erythromycin, oxitetracycline, enrofloxacin, trimethoprim/sulfamethoxazole, neomycin, and clindamycin, respectively. Coagulase-negative staphylococci (CNS) had different values for penicillin (4) and ampicillin (2), while the other antimicrobial agents had the same MIC90 values as reported for S. aureus. The MIC90 values for streptococci were 0.12, 0.25, ≤ 4, 16, ≤ 0.25, 0.5, 0.25 for penicillin, ampicillin, cephalotin, gentamicin, erythromycin, oxytetracycline and trimethoprim-sulfamethoxazole, whereas MIC90 for enterococci were 4, 4, 4, ≤ 0.5, 2, > 8 for penicillin, ampicillin, gentamicin, erythromycin, oxytetracycline and trimethoprim-sulfamethoxazole, respectively. Of 336 strains of S. aureus, 160 (47.6%) were resistant to penicillin. For 41 CNS strains, 10 (27%) presented penicillin-resistance. All the streptococcal strains were susceptible to penicillin, while 3 (7%) of the 43 enteroccocal strains were resistant. Non significant statistical differences were found between the results obtained by ADDM and broth micro-dilution for classifying bacterial isolates as susceptible or resistant according to the National Committee of Clinical Laboratory Standards.
doi:10.1186/1751-0147-43-31
PMCID: PMC1764182  PMID: 12071114
cow; mammary qland; bacteria; resistant; sensitive
4.  Antimicrobial Susceptibility and Synergy Studies of Stenotrophomonas maltophilia Isolates from Patients with Cystic Fibrosis 
Stenotrophomonas maltophilia is a newly emerging pathogen being detected with increasing frequency in patients with cystic fibrosis (CF). The impact of this multidrug-resistant organism on lung function is uncertain. The optimal treatment for S. maltophilia in CF patients is unknown. We studied the in vitro activity of ten antimicrobial agents, and conducted synergy studies by using checkerboard dilutions of eight pairs of antimicrobial agents against strains isolated from 673 CF patients from 1996 to 2001. This represents approximately 7 to 23% of the CF patients in the United States who harbor S. maltophilia annually. Doxycycline was the most active agent and inhibited 80% of 673 initial patient isolates, while trimethoprim-sulfamethoxazole inhibited only 16%. High concentrations of colistin proved more active than high concentrations of tobramycin and gentamicin. Serial isolates (n = 151) from individual patients over time (median, 290 days) showed minimal changes in resistance. Synergistic or additive activity was demonstrated by trimethoprim-sulfamethoxazole paired with ticarcillin-clavulanate (65% of strains), ciprofloxacin paired with ticarcillin-clavulanate (64% of strains), ciprofloxacin paired with piperacillin-tazobactam (59% of strains), trimethoprim-sulfamethoxazole paired with piperacillin-tazobactam (55% of strains), and doxycycline paired with ticarcillin-clavulanate (49% of strains). In all, 522 (78%) isolates were multidrug resistant (i.e., resistant to all agents in two or more antimicrobial classes) but 473 (91%) of these were inhibited by at least one antimicrobial combination (median, four; range, one to eight). To determine appropriate treatment for patients with CF, it is important to monitor the prevalence, antimicrobial susceptibility, and clinical impact of S. maltophilia in this patient population.
doi:10.1128/AAC.48.1.168-171.2004
PMCID: PMC310154  PMID: 14693535
5.  Asynchronous magnetic bead rotation (AMBR) micro-viscometer for rapid, sensitive and label-free studies of bacterial growth and drug sensitivity 
Analytical Chemistry  2012;84(12):5250-5256.
The long turnaround time in antimicrobial susceptibility testing (AST) endangers patients and encourages the administration of wide spectrum antibiotics, thus resulting in alarming increases of multi-drug resistant pathogens. A method for faster detection of bacterial proliferation presents one avenue towards addressing this global concern. We report on a label-free asynchronous magnetic bead rotation (AMBR) based viscometry method that rapidly detects bacterial growth and determines drug sensitivity by measuring changes in the suspension’s viscosity. With this platform, we observed the growth of a uropathogenic Escherichia coli isolate, with an initial concentration of 50 cells per drop, within 20 minutes; in addition, we determined the gentamicin minimum inhibitory concentration (MIC) of the E. coli isolate within 100 minutes. We thus demonstrated a label-free, micro-viscometer platform that can measure bacterial growth and drug susceptibility more rapidly, with lower initial bacterial counts than existing commercial systems, and potentially with any microbial strains.
doi:10.1021/ac300128p
PMCID: PMC3381929  PMID: 22507307
6.  Molecular Analysis of and Identification of Antibiotic Resistance Genes in Clinical Isolates of Salmonella typhi from India 
Journal of Clinical Microbiology  1998;36(6):1595-1600.
A representative sample of 21 Salmonella typhi strains isolated from cultures of blood from patients at the Christian Medical College and Hospital, Vellore, India, were tested for their susceptibilities to various antimicrobial agents. Eleven of the S. typhi strains possessed resistance to chloramphenicol (256 mg/liter), trimethoprim (64 mg/liter), and amoxicillin (>128 mg/liter), while four of the isolates were resistant to each of these agents except for amoxicillin. Six of the isolates were completely sensitive to all of the antimicrobial agents tested. All the S. typhi isolates were susceptible to cephalosporin agents, gentamicin, amoxicillin plus clavulanic acid, and imipenem. The antibiotic resistance determinants in each S. typhi isolate were encoded by one of four plasmid types. Plasmid-mediated antibiotic resistance genes were identified with specific probes in hybridization experiments; the genes responsible for chloramphenicol, trimethoprim, and ampicillin resistance were chloramphenicol acetyltransferase type I, dihydrofolate reductase type VII, and TEM-1 β-lactamase, respectively. Pulsed-field gel electrophoresis analysis of XbaI-generated genomic restriction fragments identified a single distinct profile (18 DNA fragments) for all of the resistant isolates. In comparison, six profiles, different from each other and from the resistance profile, were recognized among the sensitive isolates. It appears that a single strain containing a plasmid conferring multidrug-resistance has emerged within the S. typhi bacterial population in Vellore and has been able to adapt to and survive the challenge of antibiotics as they are introduced into clinical medicine.
PMCID: PMC104883  PMID: 9620383
7.  Repurposing Clinical Molecule Ebselen to Combat Drug Resistant Pathogens 
PLoS ONE  2015;10(7):e0133877.
Without a doubt, our current antimicrobials are losing the battle in the fight against newly-emerged multidrug-resistant pathogens. There is a pressing, unmet need for novel antimicrobials and novel approaches to develop them; however, it is becoming increasingly difficult and costly to develop new antimicrobials. One strategy to reduce the time and cost associated with antimicrobial innovation is drug repurposing, which is to find new applications outside the scope of the original medical indication of the drug. Ebselen, an organoselenium clinical molecule, possesses potent antimicrobial activity against clinical multidrug-resistant Gram-positive pathogens, including Staphylococcus, Streptococcus, and Enterococcus, but not against Gram-negative pathogens. Moreover, the activity of ebselen against Gram-positive pathogens exceeded those activities determined for vancomycin and linezolid, drugs of choice for treatment of Enterococcus and Staphylococcus infections. The minimum inhibitory concentrations of ebselen at which 90% of clinical isolates of Enterococcus and Staphylococcus were inhibited (MIC90) were found to be 0.5 and 0.25 mg/L, respectively. Ebselen showed significant clearance of intracellular methicillin-resistant S. aureus (MRSA) in comparison to vancomycin and linezolid. We demonstrated that ebselen inhibits the bacterial translation process without affecting mitochondrial biogenesis. Additionally, ebselen was found to exhibit excellent activity in vivo in a Caenorhabditis elegans MRSA-infected whole animal model. Finally, ebselen showed synergistic activities with conventional antimicrobials against MRSA. Taken together, our results demonstrate that ebselen, with its potent antimicrobial activity and safety profiles, can be potentially used to treat multidrug resistant Gram-positive bacterial infections alone or in combination with other antibiotics and should be further clinically evaluated.
doi:10.1371/journal.pone.0133877
PMCID: PMC4519285  PMID: 26222252
8.  Surveillance of antimicrobial resistance at a tertiary hospital in Tanzania 
BMC Public Health  2004;4:45.
Background
Antimicrobial resistance is particularly harmful to infectious disease management in low-income countries since expensive second-line drugs are not readily available. The objective of this study was to implement and evaluate a computerized system for surveillance of antimicrobial resistance at a tertiary hospital in Tanzania.
Methods
A computerized surveillance system for antimicrobial susceptibility (WHONET) was implemented at the national referral hospital in Tanzania in 1998. The antimicrobial susceptibilities of all clinical bacterial isolates received during an 18 months' period were recorded and analyzed.
Results
The surveillance system was successfully implemented at the hospital. This activity increased the focus on antimicrobial resistance issues and on laboratory quality assurance issues. The study identified specific nosocomial problems in the hospital and led to the initiation of other prospective studies on prevalence and antimicrobial susceptibility of bacterial infections. Furthermore, the study provided useful data on antimicrobial patterns in bacterial isolates from the hospital. Gram-negative bacteria displayed high rates of resistance to common inexpensive antibiotics such as ampicillin, tetracycline and trimethoprim-sulfamethoxazole, leaving fluoroquinolones as the only reliable oral drugs against common Gram-negative bacilli. Gentamicin and third generation cephalosporins remain useful for parenteral therapy.
Conclusion
The surveillance system is a low-cost tool to generate valuable information on antimicrobial resistance, which can be used to prepare locally applicable recommendations on antimicrobial use. The system pinpoints relevant nosocomial problems and can be used to efficiently plan further research. The surveillance system also functions as a quality assurance tool, bringing attention to methodological issues in identification and susceptibility testing.
doi:10.1186/1471-2458-4-45
PMCID: PMC526372  PMID: 15476559
9.  Efflux pump-deficient mutants as a platform to search for microbes that produce antibiotics 
Microbial Biotechnology  2015;8(4):716-725.
Pseudomonas putida DOT-T1E-18 is a strain deficient in the major antibiotic efflux pump (TtgABC) that exhibits an overall increased susceptibility to a wide range of drugs when compared with the wild-type strain. We used this strain as a platform to search for microbes able to produce antibiotics that inhibit growth. A collection of 2400 isolates from soil, sediments and water was generated and a drop assay developed to identify, via growth inhibition halos, strains that prevent the growth of DOT-T1E-18 on solid Luria–Bertani plates. In this study, 35 different isolates that produced known and unknown antibiotics were identified. The most potent inhibitor of DOT-T1E-18 growth was an isolate named 250J that, through multi-locus sequence analysis, was identified as a Pseudomonas sp. strain. Culture supernatants of 250J contain four different xantholysins that prevent growth of Gram-positive bacteria, Gram-negative and fungi. Two of the xantholysins were produced in higher concentrations and purified. Xantholysin A was effective against Bacillus, Lysinibacillus and Rhodococcus strains, and the effect against these microbes was enhanced when used in combination with other antibiotics such as ampicillin, gentamicin and kanamycin. Xantholysin C was also efficient against Gram-positive bacteria and showed an interesting antimicrobial effect against Pseudomonas strains, and a synergistic inhibitory effect with ampicillin, chloramphenicol and gentamicin.
doi:10.1111/1751-7915.12295
PMCID: PMC4476826  PMID: 26059350
10.  Treatment of uncomplicated symptomatic urinary tract infections: Resistance patterns and misuse of antibiotics 
Introduction:
Uncomplicated but symptomatic urinary tract infections (UTIs) are a common problem seen in practice. The study was undertaken to assess the most common pathogens responsible for uncomplicated symptomatic UTIs and the antimicrobial resistance pattern in a hospital in Bangalore. The study also explores the issue of antibiotic usage for these patients.
Materials and Methods:
The study was conducted in the Medicine department of a tertiary hospital in Bangalore. In all, 196 patients presented with symptoms of UTI. Bacterial growth was determined by standard microbiology techniques on freshly voided mid-steam urine samples collected from recruited patients. Patients’ demographic data, urine culture results, resistance rates to antimicrobial agents and prescribed empiric antimicrobial therapy were analyzed.
Results:
The prevalence of UTI was 32.1%; majority (67.9%) of the symptomatic did not have UTI based on culture report. Gram-negative bacteria constituted the largest group with a prevalence of 84.1% (53/63), with Escherichia coli being the most common (70%) uropathogen. Gram-negative isolates showed high level of sensitivity to amikacin (90.6%) and nitrofurantoin (77.4%). Most of the gram-positive organisms were susceptible to nitrofurantoin (70%) and gentamicin (50%). Uropathogens isolated demonstrated high resistance to cotrimoxazole, fluoroquinolones, and beta-lactam antibiotics. It was found out that 30.1% of the patients were wrongly managed of which 14.7% were over treated.
Conclusion:
UTI can be over diagnosed and over treated on the basis of clinical signs, symptoms and urine microscopy. In the era of emerging anti-microbial resistance, effective counseling and delay in antibiotic initiation or empirical therapy with a short course of nitrofurantoin is highly recommended. Empirical therapy guidelines should be updated periodically to reflect changes in antimicrobial resistance of uropathogens.
doi:10.4103/2249-4863.161342
PMCID: PMC4535106  PMID: 26288784
Anti microbial resistance; management; urinary tract infection
11.  Combinations of β-Lactam or Aminoglycoside Antibiotics with Plectasin Are Synergistic against Methicillin-Sensitive and Methicillin-Resistant Staphylococcus aureus 
PLoS ONE  2015;10(2):e0117664.
Bacterial infections remain the leading killer worldwide which is worsened by the continuous emergence of antibiotic resistance. In particular, methicillin-sensitive (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) are prevalent and the latter can be difficult to treat. The traditional strategy of novel therapeutic drug development inevitably leads to emergence of resistant strains, rendering the new drugs ineffective. Therefore, rejuvenating the therapeutic potentials of existing antibiotics offers an attractive novel strategy. Plectasin, a defensin antimicrobial peptide, potentiates the activities of other antibiotics such as β-lactams, aminoglycosides and glycopeptides against MSSA and MRSA. We performed in vitro and in vivo investigations to test against genetically diverse clinical isolates of MSSA (n = 101) and MRSA (n = 115). Minimum inhibitory concentrations (MIC) were determined by the broth microdilution method. The effects of combining plectasin with β-lactams, aminoglycosides and glycopeptides were examined using the chequerboard method and time kill curves. A murine neutropenic thigh model and a murine peritoneal infection model were used to test the effect of combination in vivo. Determined by factional inhibitory concentration index (FICI), plectasin in combination with aminoglycosides (gentamicin, neomycin or amikacin) displayed synergistic effects in 76-78% of MSSA and MRSA. A similar synergistic response was observed when plectasin was combined with β-lactams (penicillin, amoxicillin or flucloxacillin) in 87–89% of MSSA and MRSA. Interestingly, no such interaction was observed when plectasin was paired with vancomycin. Time kill analysis also demonstrated significant synergistic activities when plectasin was combined with amoxicillin, gentamicin or neomycin. In the murine models, plectasin at doses as low as 8 mg/kg augmented the activities of amoxicillin and gentamicin in successful treatment of MSSA and MRSA infections. We demonstrated that plectasin strongly rejuvenates the therapeutic potencies of existing antibiotics in vitro and in vivo. This is a novel strategy that can have major clinical implications in our fight against bacterial infections.
doi:10.1371/journal.pone.0117664
PMCID: PMC4333121  PMID: 25692771
12.  Synergistic Activity of Gentamicin with Trimethoprim or Sulfamethoxazole-Trimethoprim Against Escherichia coli and Klebsiella pneumoniae 
The effect of combinations of gentamicin with trimethoprim or sulfamethoxazole-trimethoprim against clinical isolates of Escherichia coli (11 strains) and Klebsiella pneumoniae (12 strains) was examined by using a microdilution checkerboard technique. All isolates were susceptible to each antimicrobial agent. Synergism, defined as at least a 2-log2-dilution lowering of the minimal inhibitory concentration of either antibiotic in the combination compared with the minimal inhibitory concentration of the antibiotic alone, was observed with 15 of 23 (65%) isolates tested against trimethoprim and gentamicin and 14 of 23 (61%) isolates tested against sulfamethoxazole-trimethoprim and gentamicin. A 3-log2-dilution lowering of the minimal inhibitory concentration of either antibiotic was observed in 7 of 23 (30%) trimethoprim and gentamicin trials and 3 of 23 (13%) sulfamethoxazole-trimethoprim and gentamicin trials. Antagonism was observed in 3 of 46 combination trials and only with strains of K. pneumoniae.
PMCID: PMC352530  PMID: 365085
13.  Multiresistant Uropathogenic Escherichia coli from a Region in India Where Urinary Tract Infections Are Endemic: Genotypic and Phenotypic Characteristics of Sequence Type 131 Isolates of the CTX-M-15 Extended-Spectrum-β-Lactamase-Producing Lineage 
Antimicrobial Agents and Chemotherapy  2012;56(12):6358-6365.
Escherichia coli sequence type 131 (O25b:H4), associated with the CTX-M-15 extended-spectrum beta-lactamases (ESBLs) and linked predominantly to the community-onset antimicrobial-resistant infections, has globally emerged as a public health concern. However, scant attention is given to the understanding of the molecular epidemiology of these strains in high-burden countries such as India. Of the 100 clinical E. coli isolates obtained by us from a setting where urinary tract infections are endemic, 16 ST131 E. coli isolates were identified by multilocus sequence typing (MLST). Further, genotyping and phenotyping methods were employed to characterize their virulence and drug resistance patterns. All the 16 ST131 isolates harbored the CTX-M-15 gene, and half of them also carried TEM-1; 11 of these were positive for blaOXA groups 1 and 12 for aac(6′)-Ib-cr. At least 12 isolates were refractory to four non-beta-lactam antibiotics: ciprofloxacin, gentamicin, sulfamethoxazole-trimethoprim, and tetracycline. Nine isolates carried the class 1 integron. Plasmid analysis indicated a large pool of up to six plasmids per strain with a mean of approximately three plasmids. Conjugation and PCR-based replicon typing (PBRT) revealed that the spread of resistance was associated with the FIA incompatibility group of plasmids. Pulsed-field gel electrophoresis (PFGE) and genotyping of the virulence genes showed a low level of diversity among these strains. The association of ESBL-encoding plasmid with virulence was demonstrated in transconjugants by serum assay. None of the 16 ST131 ESBL-producing E. coli strains were known to synthesize carbapenemase enzymes. In conclusion, our study reports a snapshot of the highly virulent/multiresistant clone ST131 of uropathogenic E. coli from India. This study suggests that the ST131 genotypes from this region are clonally evolved and are strongly associated with the CTX-M-15 enzyme, carry a high antibiotic resistance background, and have emerged as an important cause of community-acquired urinary tract infections.
doi:10.1128/AAC.01099-12
PMCID: PMC3497203  PMID: 23045357
14.  Using a Sequential Regimen to Eliminate Bacteria at Sublethal Antibiotic Dosages 
PLoS Biology  2015;13(4):e1002104.
We need to find ways of enhancing the potency of existing antibiotics, and, with this in mind, we begin with an unusual question: how low can antibiotic dosages be and yet bacterial clearance still be observed? Seeking to optimise the simultaneous use of two antibiotics, we use the minimal dose at which clearance is observed in an in vitro experimental model of antibiotic treatment as a criterion to distinguish the best and worst treatments of a bacterium, Escherichia coli. Our aim is to compare a combination treatment consisting of two synergistic antibiotics to so-called sequential treatments in which the choice of antibiotic to administer can change with each round of treatment. Using mathematical predictions validated by the E. coli treatment model, we show that clearance of the bacterium can be achieved using sequential treatments at antibiotic dosages so low that the equivalent two-drug combination treatments are ineffective. Seeking to treat the bacterium in testing circumstances, we purposefully study an E. coli strain that has a multidrug pump encoded in its chromosome that effluxes both antibiotics. Genomic amplifications that increase the number of pumps expressed per cell can cause the failure of high-dose combination treatments, yet, as we show, sequentially treated populations can still collapse. However, dual resistance due to the pump means that the antibiotics must be carefully deployed and not all sublethal sequential treatments succeed. A screen of 136 96-h-long sequential treatments determined five of these that could clear the bacterium at sublethal dosages in all replicate populations, even though none had done so by 24 h. These successes can be attributed to a collateral sensitivity whereby cross-resistance due to the duplicated pump proves insufficient to stop a reduction in E. coli growth rate following drug exchanges, a reduction that proves large enough for appropriately chosen drug switches to clear the bacterium.
A laboratory treatment model shows that the sequentially alternating use of two antibiotics can be optimized to outperform combination treatments at the equivalent dose.
Author Summary
So-called “cocktail” treatments are often proposed as a way of enhancing the potency of antibiotics, based on the idea that multiple drugs can synergise when used together as part of a single combined therapy. We investigated whether any other multidrug deployment strategies are as effective as—or perhaps even better than—synergistic antibiotic combinations at reducing bacterial densities. “Collateral sensitivities” between antibiotics are frequently observed; this is when measures taken by a bacterium to counter the presence of one antibiotic sensitise it to the subsequent use of another. Our approach was to see if we could exploit these sensitivities by first deploying one drug, then removing it and instead deploying another, and then repeating this process. This is not an entirely new idea, and there is a precedence for this form of treatment that has been trialled in the clinic for Helicobacter pylori infection. The idea we pursued here is an extension of “sequential treatment”; we investigated whether with two antibiotics and n rounds of treatment, if we search within the set of all possible 2n “sequential treatments”—including the two single-drug monotherapies—there might be treatments within that set that are more effective than the equivalent two-drug cocktail. Using a simple in vitro treatment model, we show that some sequential-in-time antibiotic treatments are successful under conditions that cause the failure of the cocktail treatment when implemented at the equivalent dosage.
doi:10.1371/journal.pbio.1002104
PMCID: PMC4390231  PMID: 25853342
15.  Antimicrobial-resistant Klebsiella species isolated from free-range chicken samples in an informal settlement 
Introduction
Sub-therapeutic doses of antimicrobial agents are administered routinely to poultry to aid growth and to prevent disease, with prolonged exposure often resulting in bacterial resistance. Crossover of antibiotic resistant bacteria from poultry to humans poses a risk to human health.
Material and methods
In this study, 17 chicken samples collected from a vendor operating in an informal settlement in the Cape Town Metropolitan area, South Africa were screened for antimicrobial-resistant Gram-negative bacilli using the Kirby Bauer disk diffusion assay.
Results
In total, six antibiotics were screened: ampicillin, ciprofloxacin, gentamicin, nalidixic acid, tetracycline and trimethoprim. Surprisingly, Klebsiella ozaenae was identified in 96 and K. rhinoscleromatis in 6 (n=102) of the samples tested. Interestingly, ∼40% of the isolated Klebsiella spp. showed multiple resistance to at least three of the six antibiotics tested.
Conclusions
Klebsiella ozaenae and K. rhinoscleromatis cause clinical chronic rhinitis and are almost exclusively associated with people living in areas of poor hygiene.
doi:10.5114/aoms.2012.27278
PMCID: PMC3309434  PMID: 22457672
antibiotic-resistant; poultry; resistance; Klebsiella species
16.  Metamodels for Transdisciplinary Analysis of Wildlife Population Dynamics 
PLoS ONE  2013;8(12):e84211.
Wildlife population models have been criticized for their narrow disciplinary perspective when analyzing complexity in coupled biological – physical – human systems. We describe a “metamodel” approach to species risk assessment when diverse threats act at different spatiotemporal scales, interact in non-linear ways, and are addressed by distinct disciplines. A metamodel links discrete, individual models that depict components of a complex system, governing the flow of information among models and the sequence of simulated events. Each model simulates processes specific to its disciplinary realm while being informed of changes in other metamodel components by accessing common descriptors of the system, populations, and individuals. Interactions among models are revealed as emergent properties of the system. We introduce a new metamodel platform, both to further explain key elements of the metamodel approach and as an example that we hope will facilitate the development of other platforms for implementing metamodels in population biology, species risk assessments, and conservation planning. We present two examples – one exploring the interactions of dispersal in metapopulations and the spread of infectious disease, the other examining predator-prey dynamics – to illustrate how metamodels can reveal complex processes and unexpected patterns when population dynamics are linked to additional extrinsic factors. Metamodels provide a flexible, extensible method for expanding population viability analyses beyond models of isolated population demographics into more complete representations of the external and intrinsic threats that must be understood and managed for species conservation.
doi:10.1371/journal.pone.0084211
PMCID: PMC3862810  PMID: 24349567
17.  Combination of Alpha-Melanocyte Stimulating Hormone with Conventional Antibiotics against Methicillin Resistant Staphylococcus aureus 
PLoS ONE  2013;8(9):e73815.
Our previous studies revealed that alpha-melanocyte stimulating hormone (α-MSH) is strongly active against Staphylococcus aureus (S. aureus) including methicillin resistant S. aureus (MRSA). Killing due to α-MSH occurred by perturbation of the bacterial membrane. In the present study, we investigated the in vitro synergistic potential of α-MSH with five selected conventional antibiotics viz., oxacillin (OX), ciprofloxacin (CF), tetracycline (TC), gentamicin (GM) and rifampicin (RF) against a clinical MRSA strain which carried a type III staphylococcal cassette chromosome mec (SCCmec) element and belonged to the sequence type (ST) 239. The strain was found to be highly resistant to OX (minimum inhibitory concentration (MIC) = 1024 µg/ml) as well as to other selected antimicrobial agents including α-MSH. The possibility of the existence of intracellular target sites of α-MSH was evaluated by examining the DNA, RNA and protein synthesis pathways. We observed a synergistic potential of α-MSH with GM, CF and TC. Remarkably, the supplementation of α-MSH with GM, CF and TC resulted in ≥64-, 8- and 4-fold reductions in their minimum bactericidal concentrations (MBCs), respectively. Apart from membrane perturbation, in this study we found that α-MSH inhibited ∼53% and ∼47% DNA and protein synthesis, respectively, but not RNA synthesis. Thus, the mechanistic analogy between α-MSH and CF or GM or TC appears to be the reason for the observed synergy between them. In contrast, α-MSH did not act synergistically with RF which may be due to its inability to inhibit RNA synthesis (<10%). Nevertheless, the combination of α-MSH with RF and OX showed an enhanced killing by ∼45% and ∼70%, respectively, perhaps due to the membrane disrupting properties of α-MSH. The synergistic activity of α-MSH with antibiotics is encouraging, and promises to restore the lost potency of discarded antibiotics.
doi:10.1371/journal.pone.0073815
PMCID: PMC3767696  PMID: 24040081
18.  Inhibitory activity of cranberry juice on adherence of type 1 and type P fimbriated Escherichia coli to eucaryotic cells. 
Inhibition of bacterial adherence to bladder cells has been assumed to account for the beneficial action ascribed to cranberry juice and cranberry juice cocktail in the prevention of urinary tract infections (A. E. Sobota, J. Urol. 131:1013-1016, 1984). We have examined the effect of the cocktail and juice on the adherence of Escherichia coli expressing surface lectins of defined sugar specificity to yeasts, tissue culture cells, erythrocytes, and mouse peritoneal macrophages. Cranberry juice cocktail inhibited the adherence of urinary isolates expressing type 1 fimbriae (mannose specific) and P fimbriae [specific for alpha-D-Gal(1----4)-beta-D-Gal] but had no effect on a diarrheal isolate expressing a CFA/I adhesin. The cocktail also inhibited yeast agglutination by purified type 1 fimbriae. The inhibitory activity for type 1 fimbriated E. coli was dialyzable and could be ascribed to the fructose present in the cocktail; this sugar was about 1/10 as active as methyl alpha-D-mannoside in inhibiting the adherence of type 1 fimbriated bacteria. The inhibitory activity for the P fimbriated bacteria was nondialyzable and was detected only after preincubation of the bacteria with the cocktail. Cranberry juice, orange juice, and pineapple juice also inhibited adherence of type 1 fimbriated E. coli, most likely because of their fructose content. However, the two latter juices did not inhibit the P fimbriated bacteria. We conclude that cranberry juice contains at least two inhibitors of lectin-mediated adherence of uropathogens to eucaryotic cells. Further studies are required to establish whether these inhibitors play a role in vivo.
PMCID: PMC171427  PMID: 2653218
19.  Antibiotic Resistance in Uropathogenic E. Coli Strains Isolated from Non-Hospitalized Patients in Pakistan 
Purpose: To study multidrug-resistance in Uropathogenic E. Coli (UPEC) isolated from non-hospitalized patients.
Materials and Methods: Altogether, 250 bacterial samples were collected from non-hospitalized patients. Their identifications were done on basis of Gram-staining, colony morphology, biochemical testing and PCR. Susceptibility testing was performed by using standard protocols which were recommended by CLSI.
Statistical analysis: For comparisons, statistical analysis was performed by using software, Graphpad Prism 5.0
Results: In total, 32% (n = 80) of the isolates were identified as E. Coli strains and their susceptibility patterns for different antibiotics were determined. The data indicated least resistance against tazocin [(TZP) -1.25%], amikacin [(AK) -1.8%], tigecycline [(TGC)- 2.5%] and nitrofurantoin [(F) -3.75%]. For both minocycline (MH) and sulzone (SUL), resistance rate was 5%, for gentamicin (CN), it was 16.25%, while higher resistances were observed against cephalothine [(KF)- 70%], cefotaxime [(CTX) -58.5%], ceftazidime [(CAZ)- 57.5%], cefepime [(FEP) -55%], cefuroxime and cefixime [(CXM) (CFM)- 53.75 %]. Resistance against ciprofloxacin (CIP) was 57.5%, for norfloxacine (NOR), it was 52.5% and incase of sparfloxacin (SPX), it remained 55%. High percentage of the isolates were resistant to cotrimoxazole [(SXT) -86%] and Amoxicillin [AMX-CLA (AMC)- 76%]. No resistance against meropenem (MEM) was observed.
Conclusion: Highest level of drug-resistance was observed against trimethoprim-sulfamethoxazole (TMP-SMZ) among clinical isolates of uropathogenic E. Coli collected from non-hospitalized patients.
doi:10.7860/JCDR/2014/7881.4813
PMCID: PMC4225882  PMID: 25386430
Antibiotic susceptibility; Beta-lactamase; Cotrimoxazole; E. Coli; Plasmids; UPEC
20.  Therapeutic Potential of the Antimicrobial Peptide OH-CATH30 for Antibiotic-Resistant Pseudomonas aeruginosa Keratitis 
The therapeutic potential of antimicrobial peptides (AMPs) has been evaluated in many infectious diseases. However, the topical application of AMPs for ocular bacterial infection has not been well investigated. The AMP OH-CATH30, which was identified in the king cobra, exhibits potent antimicrobial activity. In this study, we investigated the therapeutic potential of OH-CATH30 for Pseudomonas aeruginosa keratitis. Ten isolates of P. aeruginosa from individuals with keratitis were susceptible to OH-CATH30 but not to cefoperazone, ciprofloxacin, gentamicin, and levofloxacin. The microdilution checkerboard assay showed that OH-CATH30 exhibited synergistic activity with ciprofloxacin and levofloxacin against antibiotic-resistant P. aeruginosa. Meanwhile, P. aeruginosa did not develop resistance to OH-CATH30, even after exposure at 0.5× the MIC for up to 25 subcultures. Furthermore, treatment with OH-CATH30, alone or in combination with levofloxacin, significantly improved the clinical outcomes of rabbit keratitis induced by antibiotic-resistant P. aeruginosa. Taken together, our data indicate that the topical application of OH-CATH30 is efficacious against drug-resistant P. aeruginosa keratitis. In addition, our study highlights the potential application of AMPs in treating ocular bacterial infections.
doi:10.1128/AAC.00095-14
PMCID: PMC4068471  PMID: 24637683
21.  Klebsiella species: antimicrobial susceptibilities, bactericidal kinetics, and in vitro inactivation of beta-lactam agents. 
In vitro properties of 19 antimicrobial agents were tested with 56 isolates of Klebsiella spp. The aminoglycosides and the new beta-lactam compounds cefotaxime and moxalactam were the most inhibitory drugs tested. Chloramphenicol, tetracycline, trimethoprim, and trimethoprim-sulfamethoxazole were moderately active, whereas piperacillin, mezlocillin, and furazlocillin were ineffective against 25% of the isolates. Gentamicin was the only agent tested that was uniformly bactericidal in time-kill experiments with drug concentrations of four times the minimal inhibitory concentration. In combination studies with gentamicin, moxalactam and furazlocillin each increased the rate of bacterial killing for three of five isolates as compared with gentamicin alone, whereas chloramphenicol significantly retarded the rate of bacterial killing for the same number of strains. Furazlocillin was completely inactivated after 24 h of incubation with each of five selected strains. The inactivation of moxalactam, cefoxitin, and cephalothin was 36, 56, and 72%, respectively. In all instances in which these four agents were inactivated to levels below the minimal bactericidal concentration, there was accelerated growth after initial inhibition. However, regrowth also occurred in three instances in which drug levels were higher than the minimal bactericidal concentration. Retesting after drug exposure revealed a 4- to 32-fold rise in the minimal inhibitory concentration and minimal bactericidal concentration in two of these isolates.
PMCID: PMC352982  PMID: 7235676
22.  Phenylbutyrate Is Bacteriostatic against Mycobacterium tuberculosis and Regulates the Macrophage Response to Infection, Synergistically with 25-Hydroxy-Vitamin D₃ 
PLoS Pathogens  2015;11(7):e1005007.
Adjunctive vitamin D treatment for pulmonary tuberculosis enhances resolution of inflammation but has modest effects on bacterial clearance. Sodium 4-phenylbutyrate (PBA) is in clinical use for a range of conditions and has been shown to synergise with vitamin D metabolites to upregulate cathelicidin antimicrobial peptide (CAMP) expression. We investigated whether clinically attainable plasma concentrations of PBA (0.4-4mM) directly affect Mycobacterium tuberculosis (Mtb) growth and human macrophage and PBMC response to infection. We also tested the ability of PBA to enhance the immunomodulatory actions of the vitamin D metabolite 25(OH)D3 during infection and synergistically inhibit intracellular Mtb growth. PBA inhibited Mtb growth in broth with an MIC99 of 1mM, which was reduced to 0.25mM by lowering pH. During human macrophage infection, PBA treatment restricted Mtb uptake, phagocytic receptor expression and intracellular growth in a dose-dependent manner. PBA independently regulated CCL chemokine secretion and induced expression of the antimicrobial LTF (lactoferrin), the anti-inflammatory PROC (protein C) and multiple genes within the NLRP3 inflammasome pathway. PBA co-treatment with 25(OH)D3 synergistically modulated expression of numerous vitamin D-response genes, including CAMP, CYP24A1, CXCL10 and IL-37. This synergistic effect was dependent on MAPK signalling, while the effect of PBA on LTF, PROC and NLRP3 was MAPK-independent. During PBA and 25(OH)D3 co-treatment of human macrophages, in the absence of exogenous proteinase 3 (PR3) to activate cathelicidin, Mtb growth restriction was dominated by the effect of PBA, while the addition of PR3 enhanced growth restriction by 25(OH)D3 and PBA co-treatment. This suggests that PBA augments vitamin D–mediated cathelicidin-dependent Mtb growth restriction by human macrophages and independently induces antimicrobial and anti-inflammatory action. Therefore through both host-directed and bacterial-directed mechanisms PBA and vitamin D may prove an effective combinatorial adjunct therapy for tuberculosis to both resolve immunopathology and enhance bacterial clearance.
Author Summary
Tuberculosis (TB) is the world’s leading bacterial cause of death. Effective treatment currently requires a minimum of 4 drugs taken for at least 6 months. While these drugs kill the TB causing bacteria (Mtb), they do not directly resolve the inmmunopathology associated with morbidity. Immunomodulatory agents that not only enhance an individual’s ability to kill Mtb but also help heal lung pathology could be used as adjuncts to current therapies to improve treatment outcome. Phenylbutyrate (PBA) has been in clinical use for more than 30 years to treat a range of conditions. It has also been shown to synergise with vitamin D to induce cellular production of the anti-Mtb peptide, cathelicidin. We investigated whether PBA and vitamin D synergistically kill Mtb in human macrophages and whether PBA has any independent effect on macrophages and Mtb. At concentrations that are achieved in plasma clinically, PBA inhibited Mtb growth. PBA also inhibited growth of Mtb in human macrophages via a cell-dependent mechanism, inducing the inflammasome pathway and antimicrobial lactoferrin. PBA also synergistically enhanced macrophage response to vitamin D and co-treatment further inhibited Mtb growth, when synergistically-induced cathelicidin was activated. PBA and vitamin D may therefore prove an effective combinatorial adjunct therapy for tuberculosis.
doi:10.1371/journal.ppat.1005007
PMCID: PMC4489717  PMID: 26133770
23.  Comparison of Clinical Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing guidelines for the interpretation of antibiotic susceptibility at a University teaching hospital in Nairobi, Kenya: a cross-sectional study 
Background
The Clinical Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines are the most popular breakpoint guidelines used in antimicrobial susceptibility testing worldwide. The EUCAST guidelines are freely available to users while CLSI is available for non-members as a package of three documents for US $500 annually. This is prohibitive for clinical microbiology laboratories in resource poor settings. We set out to compare antibiotic susceptibility determined by the two guidelines to determine whether adoption of EUCAST guidelines would significantly affect our susceptibility patterns.
Methods
We reviewed minimum inhibitory concentrations (MIC) of various antibiotics routinely reported for Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) isolates from an automated microbiology identification system (VITEK-2) at the Aga Khan University Hospital Nairobi’s Pathology department. These MICs were then analyzed using both CLSI 2015 and EUCAST 2015 guidelines and classified as resistant, intermediate or susceptible. We compared the susceptibility and agreement between the CLSI and EUCAST categorizations.
Results
Susceptibility data from a total of 5165 E. coli, 1103 S. aureus and 532 P. aeruginosa isolates were included. The concordance rates of the two guidelines for E. coli, S. aureus and P. aeruginosa ranged from 78.2 to 100 %, 94.6 to 100 % and 89.1 to 95.5 % respectively. The kappa statistics for E. coli MICs revealed perfect agreement between CLSI and EUCAST for cefotaxime, ceftriaxone and trimethoprim–sulfamethoxazole, almost perfect agreement for ampicillin, ciprofloxacin, cefuroxime, gentamicin and ceftazidime, substantial agreement for meropenem, moderate agreement for cefepime and amoxicillin-clavulanate, fair agreement for nitrofurantoin and poor agreement for amikacin. For S. aureus the kappa statistics revealed perfect agreement for penicillin, trimethoprim–sulfamethoxazole, levofloxacin, oxacillin, linezolid and vancomycin, almost perfect agreement for clindamycin, erythromycin and tetracycline and moderate agreement for gentamicin. For P. aeruginosa the kappa analysis revealed moderate to almost perfect agreement for all the anti-pseudomonal antibiotics.
Conclusion
The results show comparable antibiotic susceptibility patterns between CLSI and EUCAST breakpoints. Given that EUCAST guidelines are freely available, it makes it easier for laboratories in resource poor settings to have an updated and readily available reference for interpreting antibiotic susceptibilities.
doi:10.1186/s12941-016-0135-3
PMCID: PMC4827198  PMID: 27068515
European Committee on Antimicrobial Susceptibility Testing (EUCAST); Clinical Laboratory Standards Institute (CLSI); Antimicrobial susceptibility testing (AST); Minimum inhibitory concentrations (MIC)
24.  Antibiotic-specific differences in the response of Staphylococcus aureus to treatment with antimicrobials combined with manuka honey 
Skin infections caused by antibiotic resistant Staphylococcus aureus are a significant health problem worldwide; often associated with high treatment cost and mortality rate. Complex natural products like New Zealand (NZ) manuka honey have been revisited and studied extensively as an alternative to antibiotics due to their potent broad-spectrum antimicrobial activity, and the inability to isolate honey-resistant S. aureus. Previous studies showing synergistic effects between manuka-type honeys and antibiotics have been demonstrated against the growth of one methicillin-resistant S. aureus (MRSA) strain. We have previously demonstrated strong synergistic activity between NZ manuka-type honey and rifampicin against growth and biofilm formation of multiple S. arueus strains. Here, we have expanded our investigation using multiple S. aureus strains and four different antibiotics commonly used to treat S. aureus-related skin infections: rifampicin, oxacillin, gentamicin, and clindamycin. Using checkerboard microdilution and agar diffusion assays with S. aureus strains including clinical isolates and MRSA we demonstrate that manuka-type honey combined with these four antibiotics frequently produces a synergistic effect. In some cases when synergism was not observed, there was a significant enhancement in antibiotic susceptibility. Some strains that were highly resistant to an antibiotic when present alone become sensitive to clinically achievable concentrations when combined with honey. However, not all of the S. aureus strains tested responded in the same way to these combinational treatments. Our findings support the use of NZ manuka-type honeys in clinical treatment against S. aureus-related infections and extend their potential use as an antibiotic adjuvant in combinational therapy. Our data also suggest that manuka-type honeys may not work as antibiotic adjuvants for all strains of S. aureus, and this may help determine the mechanistic processes behind honey synergy.
doi:10.3389/fmicb.2014.00779
PMCID: PMC4307217  PMID: 25674077
Medihoney; Staphylococcus aureus; manuka; antibiotic; synergy
25.  Synergy of fosmidomycin (FR-31564) and other antimicrobial agents. 
Fosmidomycin (FR-31564), a phosphonic acid derivative, was combined with cefazolin, cephalexin, ampicillin, carbenicillin, ticarcillin, gentamicin, and trimethoprim. Synergy between fosmidomycin and penicillins or cephalosporins was found for 37 to 52% of the Enterobacteriaceae tested. Synergy with trimethoprim was found against 55% of bacteria isolated, but only 17% of the strains showed synergy between formidomycin and gentamicin. Synergy between fosmidomycin and ticarcillin was shown for 35% of the Pseudomonas isolates. Cefazolin-, ampicillin-, and gentamicin-resistant isolates of various species were synergistically inhibited by fosmidomycin, as were ticarcillin- and gentamicin-resistant isolates. Antagonism was not encountered. This study illustrates another example of synergistic activity of compounds which attack different mechanisms in bacterial cells.
PMCID: PMC183793  PMID: 7181471

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