Prevalence and Antimicrobial Susceptibility Patterns of Bacteria from Milkmen and Cows with Clinical Mastitis in and around Kampala, Uganda

<div><p>Background</p><p>Identification of pathogens associated with bovine mastitis is helpful in treatment and management decisions. However, such data from sub-Saharan Africa is scarce. Here we describe the distribution and antimicrobial susceptibility patterns of bacteria from cows with clinical mastitis in Kampala, Uganda. Due to high concern of zoonotic infections, isolates from milkmen are also described.</p><p>Methodology/Principal Findings</p><p>Ninety seven milk samples from cows with clinical mastitis and 31 nasal swabs from milkmen were collected (one sample per cow/human). Fifty eight (60%) Gram-positive isolates namely Staphylococci (21), Enterococci (16), Streptococci (13), Lactococci (5), Micrococci (2) and Arcanobacteria (1) were detected in cows; only one grew <i>Staphylococcus aureus</i>. Furthermore, 24 (25%) coliforms namely <i>Escherichia coli</i> (12), <i>Klebsiella oxytoca</i> (5), <i>Proteus vulgaris</i> (2), <i>Serratia</i> (2), <i>Citrobacter</i> (1), <i>Cedecea</i> (1) and <i>Leclercia</i> (1) were identified. From humans, 24 Gram-positive bacteria grew, of which 11 were Staphylococci (35%) including four <i>Staphylococcus aureus</i>. Upon susceptibility testing, methicillin-resistant coagulase-negative staphylococci (CoNS) were prevalent; 57%, 12/21 in cows and 64%, 7/11 in humans. However, methicillin-resistant <i>Staphylococcus aureus</i> was not detected. Furthermore, methicillin and vancomycin resistant CoNS were detected in cows (<i>Staphylococcus hominis</i>, <i>Staphylococcus lugdunensis</i>) and humans (<i>Staphylococcus scuiri</i>). Also, vancomycin and daptomycin resistant Enterococci (<i>Enterococcus faecalis</i> and <i>Enterococcus faecium,</i> respectively) were detected in cows. Coliforms were less resistant with three pan-susceptible isolates. However, multidrug resistant <i>Klebsiella</i>, <i>Proteus</i>, <i>Serratia, Cedecea</i>, and <i>Citrobacter</i> were detected. Lastly, similar species grew from human and bovine samples but on genotyping, the isolates were found to be different. Interestingly, human and bovine <i>Staphylococcus aureus</i> were genetically similar (spa-CC435, spa-type t645 corresponding to ST121) but with different susceptibility patterns.</p><p>Conclusions/Significance</p><p>CoNS, Enterococci, Streptococci, and <i>Escherichia coli</i> are the predominant pathogens associated with clinical bovine-mastitis in Kampala, Uganda. Multidrug resistant bacteria are also prevalent. While similar species occurred in humans and cows, transmission was not detected.</p></div

Antimicrobial resistance among staphylococci from cows (panel A) and milkmen (panel B).

<p>Details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063413#pone.0063413.s001" target="_blank">Tables S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063413#pone.0063413.s002" target="_blank">S2</a>.</p

Distinct patterns among staphylococci (panel A), enterococci (panel B) and streptococci (panel C) following RAPD genotyping.

<p>One isolate per lane.</p

Antimicrobial resistance patterns among coliforms (n = 24).

<p>AMP, Ampicillin; AMO; Amoxicillin-Clavulanate; SXT, trimethopprim-sulfamethoxazole; COL, Colistin; IMP, imipenem; CEF, Cefoxitine; CFT, Cefotaxim; CEP, Cephalothin; CFU, Cefuroxime; CFP, Cefepime; AZT, Aztreonam; ERY, Erythromycin; NTR, Nitrofurantoin; PIP, Piperacillin-Tazobactum; ERT, Ertapenem.</p><p>In boldface type are isolates found to be multi-drug resistant (MDR).</p

Frequency and patterns of resistance conferring mutations in <i>M</i>. <i>tuberculosis</i> isolates that were resistant to rifampicin (n = 45) and isoniazid (n = 50).

<p>Frequency and patterns of resistance conferring mutations in <i>M</i>. <i>tuberculosis</i> isolates that were resistant to rifampicin (n = 45) and isoniazid (n = 50).</p

Frequency of rifampicin/isoniazid-resistance conferring mutations in the circulating <i>M</i>. <i>tuberculosis</i> genotypes/sub-lineages in Uganda (2009–2011).

<p>Frequency of rifampicin/isoniazid-resistance conferring mutations in the circulating <i>M</i>. <i>tuberculosis</i> genotypes/sub-lineages in Uganda (2009–2011).</p

Rhomboids of Mycobacteria: Characterization Using an <em>aarA</em> Mutant of <em>Providencia stuartii</em> and Gene Deletion in <em>Mycobacterium smegmatis</em>

<div><h3>Background</h3><p>Rhomboids are ubiquitous proteins with unknown roles in mycobacteria. However, bioinformatics suggested putative roles in DNA replication pathways and metabolite transport. Here, mycobacterial rhomboid-encoding genes were characterized; first, using the <em>Providencia stuartii</em> null-rhomboid mutant and then deleted from <em>Mycobacterium smegmatis</em> for additional insight in mycobacteria.</p> <h3>Methodology/Principal Findings</h3><p>Using in silico analysis we identified in <em>M. tuberculosis</em> genome the genes encoding two putative rhomboid proteins; Rv0110 (referred to as “rhomboid protease 1”) and Rv1337 (“rhomboid protease 2”). Genes encoding orthologs of these proteins are widely represented in all mycobacterial species. When transformed into <em>P. stuartii</em> null-rhomboid mutant (Δ<em>aarA</em>), genes encoding mycobacterial orthologs of “rhomboid protease 2” fully restored AarA activity (AarA is the rhomboid protein of <em>P. stuartii</em>). However, most genes encoding mycobacterial “rhomboid protease 1” orthologs did not. Furthermore, upon gene deletion in <em>M. smegmatis</em>, the ΔMSMEG_4904 single mutant (which lost the gene encoding MSMEG_4904, orthologous to Rv1337, “rhomboid protease 2”) formed the least biofilms and was also more susceptible to ciprofloxacin and novobiocin, antimicrobials that inhibit DNA gyrase. However, the ΔMSMEG_5036 single mutant (which lost the gene encoding MSMEG_5036, orthologous to Rv0110, “rhomboid protease 1”) was not as susceptible. Surprisingly, the double rhomboid mutant ΔMSMEG_4904–ΔMSMEG_5036 (which lost genes encoding both homologs) was also not as susceptible suggesting compensatory effects following deletion of both rhomboid-encoding genes. Indeed, transforming the double mutant with a plasmid encoding MSMEG_5036 produced phenotypes of the ΔMSMEG_4904 single mutant (i.e. susceptibility to ciprofloxacin and novobiocin).</p> <h3>Conclusions/Significance</h3><p>Mycobacterial rhomboid-encoding genes exhibit differences in complementing <em>aarA</em> whereby it's only genes encoding “rhomboid protease 2” orthologs that fully restore AarA activity. Additionally, gene deletion data suggests inhibition of DNA gyrase by MSMEG_4904; however, the ameliorated effect in the double mutant suggests occurrence of compensatory mechanisms following deletion of genes encoding both rhomboids.</p> </div

Growth inhibition assays.

<p>Depicted are rhomboid mutants (Δ4904, Δ5036 and Δ4904Δ5036) and the wild type (WT) cultured in media with 0.1 µg/ml ciprofloxacin <b>(panel A)</b>; 60 µg/ml novobiocin <b>(panel B)</b>; 100 µg/ml isoniazid <b>(INH, panel C)</b>; and 0.5 µg/ml kanamycin <b>(panel D)</b>. The Δ4904 single mutant (Δ4904) was inhibited by ciprofloxacin (0.1 µg/ml) and novobiocin (60 µg ml⁻¹). In-set are similar data on solid media (7H10) showing that the Δ4904 single mutant (Δ4904) struggles to grow in presence of 0.1 µg ml⁻¹ ciprofloxacin and 60 µg ml⁻¹ novobiocin. Each data point was from an average of four experiments.</p

Figure 2

<p><b>Panels A and B:</b> Wild type <i>M. smegmatis</i> (WT) and rhomboid mutants (Δ4904 single, Δ5036 single and Δ4904Δ5036 double) cultured at 37°C (<b>Panel A</b>) and 42°C (<b>Panel B</b>), showing no difference in growth patterns. <b>Panel C:</b> Colony magnification showing differences in morphology between mutants (Δ4904, Δ5036 and Δ4904Δ5036) and the wild type. <b>Panel D: </b><i>M. smegmatis</i> single rhomboid mutants were inefficient at biofilm formation. The Δ4904 single mutant (Δ4904) formed the least biofilms while the double mutant (Δ4904Δ5036) formed more biofilms than the single mutants. Each data point was from an average of four experiments.</p